Compositions for the treatment and diagnosis of immune disorders

ABSTRACT

The present invention relates to methods and compositions for the treatment and diagnosis of immune disorders, especially T helper lymphocyte-related disorders. For example, genes which are differentially expressed within and among T helper (TH) cells and TH cell subpopulations, which include, but are not limited to TH0, TH1 and TH2 cell subpopulations are identified. Genes are also identified via the ability of their gene products to interact with gene products involved in the differentiation, maintenance and effector function of such TH cells and TH cell subpopulations. The genes identified can be used diagnostically or as targets for therapeutic intervention. In this regard, the present invention provides methods for the identification and therapeutic use of compounds as treatments of immune disorders, especially TH cell subpopulation-related disorders. Additionally, methods are provided for the diagnostic evaluation and prognosis of TH cell subpopulation-related disorders, for the identification of subjects exhibiting a predisposition to such conditions, for monitoring patients undergoing clinical evaluation for the treatment of such disorders, and for monitoring the efficacy of compounds used in clinical trials.

This application is a Divisional of application Ser. No. 08/487,748,filed Jun. 7, 1995, now U.S. Pat. No. 5,721,351 which is aContinuation-In-Part of application Ser. No. 08/398,633, filed Mar. 3,1995, the entire contents of both of which are incorporated herein byreference in their entirety.

1. INTRODUCTION

The present invention relates to methods and compositions for thetreatment and diagnosis of immune disorders, especially Tlymphocyte-related disorders, including, but not limited to, chronicinflammatory diseases and disorders, such as Crohn's disease, reactivearthritis, including Lyme disease, insulin-dependent diabetes,organ-specific autoimmunity, including multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease, contact dermatitis, psoriasis, graftrejection, graft versus host disease, sarcoidosis, atopic conditions,such as asthma and allergy, including allergic rhinitis,gastrointestinal allergies, including food allergies, eosinophilia,conjunctivitis, glomerular nephritis, certain pathogen susceptibilitiessuch as helminthic (e.g., leishmaniasis) and certain viral infections,including HIV, and bacterial infections, including tuberculosis andlepromatous leprosy. For example, genes which are differentiallyexpressed within and among T helper (TH) cells and TH cellsubpopulations, which include, but are not limited to TH0, TH1 and TH2cell subpopulations are identified. Genes are also identified via theability of their gene products to interact with gene products involvedin the differentiation, maintenance and effector function of such THcells and TH cell subpopulations. The genes identified can be useddiagnostically or as targets for therapeutic intervention. In thisregard, the present invention provides methods for the identificationand therapeutic use of compounds as treatments of immune disorders,especially TH cell subpopulation-related disorders. Additionally,methods are provided for the diagnostic evaluation and prognosis of THcell subpopulation-related disorders, for the identification of subjectsexhibiting a predisposition to such conditions, for monitoring patientsundergoing clinical evaluation for the treatment of such disorders, andfor monitoring the efficacy of compounds used in clinical trials.

2. BACKGROUND OF THE INVENTION

Two distinct types of T lymphocytes are recognized: CD8⁺ cytotoxic Tlymphocytes (CTLs) and CD4⁺ helper T lymphocytes (TH cells). CTLsrecognize and kill cells which display foreign antigens of theirsurfaces. CTL precursors display T cell receptors that recognizeprocessed peptides derived from foreign proteins, in conjunction withclass I MHC molecules, on other cell surfaces. This recognition processtriggers the activation, maturation and proliferation of the precursorCTLs, resulting in CTL clones capable of destroying the cells exhibitingthe antigens recognized as foreign.

TH cells are involved in both humoral and cell-mediated forms ofeffector immune responses. With respect to the humoral, or antibody,immune response, antibodies are produced by B lymphocytes throughinteractions with TH cells. Specifically, extracellular antigens areendocytosed by antigen-presenting cells (APCs), processed, and presentedpreferentially in association with class II major histocompatibilitycomplex (MHC) molecules to CD4⁺ class II MHC-restricted TH cells. TheseTH cells in turn activate B lymphocytes, resulting in antibodyproduction.

The cell-mediated, or cellular, immune response, functions to neutralizemicrobes which inhabit intracellular locations. Foreign antigens, suchas, for example, viral antigens, are synthesized within infected cellsand presented on the surfaces of such cells in association with class IMHC molecules. This, then, leads to the stimulation of the CD8⁺ class IMHC-restricted CTLs.

Some agents, such as mycobacteria, which cause tuberculosis and leprosy,are engulfed by macrophages and processed in vacuoles containingproteolytic enzymes and other toxic substances. While these macrophagecomponents are capable of killing and digesting most microbes, agentssuch as mycobacteria survive and multiply. The agents' antigens areprocessed, though, by the macrophages and presented preferentially inassociation with class II MHC molecules to CD4⁺ class II MHC-restrictedTH cells, which become stimulated to secrete interferon-γ, which, inturn, activates macrophages. Such activation results in the cells'exhibiting increased bacteriocidal ability.

TH cells are composed of at least two distinct subpopulations, termedTH1 and TH2 cell subpopulations. Evidence suggests that TH1 and TH2subtypes represent extremely polarized populations of TH cells. Whilesuch subpopulations were originally discovered in murine systems(reviewed in Mosmann, T. R. and Coffman, R. L., 1989, Ann. Rev. Immunol.7:145), the existence of TH1- and TH2-like subpopulations has also beenestablished in humans (Del Prete, A. F. et al., 1991, J. Clin. Invest.88:346; Wiernenga, E. A. et al., 1990, J. Imm. 144:4651; Yamamura, M. etal., 1991, Science 254:277; Robinson, D. et al., 1993, J. Allergy Clin.Imm. 92:313). While TH1-like and TH2-like cells can represent the mostextremely polarized TH cell subpopulations, other TH cellsubpopulations, such as TH0 cells (Firestein, G. S. et al., 1989, J.Imm. 143:518), which represent TH cells which have characteristics ofTH1 and TH2 cell subpopulations.

TH1-like and TH2-like cells appear to function as part of the differenteffector functions of the immune system (Mosmann, T. R. and Coffmann, R.L., 1989, Ann. Rev. Imm. 7:145). Specifically, TH1-like cells direct thedevelopment of cell-mediated immunity, triggering phagocyte-mediatedhost defenses, and are associated with delayed hypersensitivity.Accordingly, infections with intracellular microbes tend to induceTH1-type responses. TH2 cells drive humoral immune responses, which areassociated with, for example, defenses against certain helminthicparasites, and are involved in antibody and allergic responses.

It has been noted that the ability of the different TH cell types todrive different immune effector responses is due to the exclusivecombinations of cytokines which are expressed within a particular THcell subpopulation. For example, TH1 cells are known to secreteinterleukin-2 (IL-2), interferon-γ (IFN-γ), and lymphotoxin, while TH2cells secrete interleukin-4 (IL-4), interleukin-5 (IL-5), andinterleukin-10 (IL-10).

It is thought that TH1 and TH2 subpopulations arise from a common naiveprecursor (referred to as THP). For example, naive CD4⁺ cells from micewhich express a single transgenic T cell receptor can be induced todevelop into either the TH1 or TH2 cell type. The conditions of antigenstimulation, including the nature and amount of antigen involved, thetype of antigen-presenting cells, and the type of hormone and cytokinemolecules present seem to all represent determinants of the pattern ofTH1 versus TH2 differentiation, with, perhaps, the decisive rolebelonging to the cytokines present. With such a complex series ofpossible determinants, a full accounting of the exact factors importantin driving TH1 or TH2 differentiation are, as yet largely unknown.

Further, it has recently been noted that, in addition to CD4⁺ TH cells,CD8⁺ CTLs can, under certain conditions, also exhibit TH1-like orTH2-like cytokine profiles (Seder, R. A. et al., 1995, J. Exp. Med.181:5-7; Manetti, R. et al., 1994, J. Exp. Med. 180:2407-2411; Maggi, E.et al., 1994, J. Exp. Med. 180:489-495). While the precise functionalrole of such CD8⁺ TH-like cells is currently unknown, these cellsubpopulations appear to have great relevance to immune responsesagainst infectious agents such as viruses and intracellular parasites.

Once TH1 and TH2 subpopulations are expanded, the cell types tend tonegatively regulate one another through the actions of cytokines uniqueto each. For example, TH1-produced IFN-γ negatively regulates TH2 cells,while TH2-produced IL-10 negatively regulates TH1 cells. Moreover,cytokines produced by TH1 and TH2 antagonize the effector functions ofone another (Mosmann, T. R. and Moore, 1991, Immunol. Today 12:49).

Failure to control or resolve an infectious process often results froman inappropriate, rather than an insufficient immune response, and canunderlie a variety of distinct immunological disorders. Such disorderscan include, for example, atopic conditions (i.e., IgE-mediated allergicconditions) such as asthma, allergy, including allergic rhinitis,dermatitis, including psoriasis, pathogen susceptibilities, chronicinflammatory disease, organ-specific autoimmunity, graft rejection andgraft versus host disease. For example, nonhealing forms of human andmurine leishmaniasis result from strong but counterproductiveTH2-like-dominated immune responses. Lepromatous leprosy also appears tofeature a prevalent, but inappropriate, TH2-like response.

It is possible that another example can be HIV infection. Here, it hasbeen suggested that a drop in the ratio of TH1-like cells to other THcell subpopulations can play a critical role in the progression towarddisease symptoms. Further, it has been noted that, at least in vitro,TH2-like clones appear to be more efficient supporters of HIV viralreplication than TH1-like clones.

Further, while TH1-mediated inflammatory responses to many pathogenicmicroorganisms are beneficial, such responses to self antigens areusually deleterious. It has been suggested that the preferentialactivation of TH1-like responses is central to the pathogenesis of suchhuman inflammatory autoimmune diseases as multiple sclerosis andinsulin-dependent diabetes. For example, TH1-type cytokines predominatein the cerebrospinal fluid of patients with multiple sclerosis,pancreases of insulin-dependent diabetes patients, thyroid glands ofHashimoto's thyroiditis, and gut of Crohn's disease patients, suggestingthat such patients mount a TH1-like, not a TH2-like, response to theantigen(s) involved in the etiopathogenesis of such disorders.

A primary goal, for both diagnostic and therapeutic reasons, therefore,would be the ability to identify, isolate and/or target members of aparticular TH cell subpopulation. The ability to identify those geneswhich are differentially expressed within and/or among such TH cellsubpopulations is required to achieve such a goal. To date,investigations have focused on the expression of a limited number ofspecific known cytokines and cytokine receptors in the TH cellpopulation. Cytokines, however, exert effects on cell types in additionto specific TH cell subpopulations, i.e., exhibit a variety ofpleiotropic effects. It would be beneficial, therefore, to identifyreliable markers (e.g., gene sequences) of TH cell subpopulations whoseeffects are TH cell subpopulation specific, e.g., which, unlike secretedcytokines, are TH cell subpopulation specific.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of immune disorders, especially T helper (TH) cell and THcell-like related disorders. First, genes are identified and describedwhich are differentially expressed within and among TH cells and TH cellsubpopulations. Second, genes are identified and described which aredifferentially expressed within TH cell subpopulations in TH cellsubpopulation-related disorders. The modulation of the expression of theidentified genes and/or the activity of the identified gene products canbe utilized therapeutically to ameliorate immune disorder symptoms andto modulate TH cell responsiveness, for example, responsiveness toantigen. Further, the identified genes and/or gene products can be usedto diagnose individuals exhibiting or predisposed to such immunedisorders. Still further, the identified genes and/or gene products canbe used to detect TH cell responsiveness, for example, responsiveness toantigen.

"Differential expression," as used herein, refers to both quantitativeas well as qualitative differences in the genes' temporal and/orcellular expression patterns within and among the TH cellsubpopulations. Differentially expressed genes can represent"fingerprint genes" and/or "target genes".

"Fingerprint gene," as used herein, refers to a differentially expressedgene whose expression pattern can be utilized as part of a prognostic ordiagnostic evaluation of immune disorders, e.g., TH cell-relateddisorders, or which, alternatively, can be used in methods foridentifying compounds useful in the treatment of such disorders. Forexample, the effect of the compound on the fingerprint gene expressionnormally displayed in connection with the disorder can be used toevaluate the efficacy of the compound as a treatment for such adisorder, or may, additionally, be used to monitor patients undergoingclinical evaluation for the treatment of such disorders.

"Fingerprint pattern," as used herein, refers to the pattern generatedwhen the expression pattern of a series (which can range from two up toall the fingerprint genes which exist for a given state) of fingerprintgenes is determined. A fingerprint pattern can be used in the samediagnostic, prognostic, and compound identification methods as theexpression of a single fingerprint gene.

"Target gene," as used herein, refers to a differentially expressed geneinvolved in immune disorders, e.g., TH cell related disorders, such thatmodulation of the level of target gene expression or of a target geneproduct activity can act to ameliorate the immune disorder. Compoundsthat modulate target gene expression or activity of the target geneproduct can be used in the treatment of immune disorders.

Further, "pathway genes" are defined via the ability of their geneproducts to interact with gene products involved in TH cellsubpopulation-related disorders and/or to interact with gene productswhich are involved in the differentiation and effector function of theTH cell subpopulations. Pathway genes can also exhibit target geneand/or fingerprint gene characteristics.

Although the target, fingerprint and/or pathway genes described hereincan be differentially expressed within and/or among TH cellsubpopulations, and/or can interact with TH cell subpopulation geneproducts, the genes can also be involved in mechanisms important toadditional immune processes.

The present invention includes the products of such fingerprint, target,and pathway genes, as well as antibodies to such gene products.Furthermore, the engineering and use of cell- and animal-based models ofTH cell subpopulation-related disorders to which such gene products cancontribute, are also described.

The present invention also relates to methods for prognostic anddiagnostic evaluation of various TH cell subpopulation-relateddisorders, and for the identification of subjects who are predisposed tosuch disorders. Furthermore, the invention provides methods forevaluating the efficacy of drugs for immune disorders, and monitoringthe progress of patients involved in clinical trials for the treatmentof such disorders.

The TH cell subpopulation-related disorders described herein caninclude, for example, TH1 or TH1-like related disorders or can,alternatively, include TH2 or TH2-like related disorders. Examples ofTH1 or TH1-like related disorders include chronic inflammatory diseasesand disorders, such as Crohn's disease, reactive arthritis, includingLyme disease, insulin-dependent diabetes, organ-specific autoimmunity,including multiple sclerosis, Hashimoto's thyroiditis and Grave'sdisease, contact dermatitis, psoriasis, graft rejection, graft versushost disease and sarcoidosis. Examples of TH2 or TH2-like relateddisorders include atopic conditions, such as asthma and allergy,including allergic rhinitis, gastrointestinal allergies, including foodallergies, eosinophilia, conjunctivitis, glomerular nephritis, certainpathogen susceptibilities such as helminthic (e.g., leishmaniasis) andcertain viral infections, including HIV, and bacterial infections,including tuberculosis and lepromatous leprosy.

It is further contemplated that the methods and compositions describedherein can be utilized in the prognostic and diagnostic evaluation ofdisorders involving other immune cells, including CD8⁺ CTLs, exhibitingTH-like cell subpopulation gene expression patterns and/or activity. Itis still further contemplated that the methods and compositionsdescribed herein can be utilized in the amelioration of symptomsstemming from disorders involving such immune cells, especially suchCD8⁺ CTLs, which exhibit TH-like cell subpopulation gene expressionpatterns and/or activity.

The invention further provides methods for the identification ofcompounds which modulate the expression of genes or the activity of geneproducts involved in TH cell subpopulation-related disorders andprocesses relevant to the differentiation, maintenance and/or effectorfunction of the subpopulations. Still further, the present inventionprovides methods for the treatment of TH cell subpopulation-relateddisorders which can, for example, involve the administration of suchmodulatory compounds to individuals exhibiting TH cellsubpopulation-related disorder symptoms or tendencies. Additionally,treatment can result in the stimulation or depletion of one or more ofthe TH cell subpopulations.

"Stimulation", as used herein, can refer to an effective increase in thenumber of cells belonging to a TH cell subpopulation, via, for example,the proliferation of such TH cell subpopulation cells. The term can alsorefer to an increase in the activity of cells belonging to a TH cellsubpopulation, as would be evidenced, for example, by a per cellincrease in the expression of the TH cell subpopulation-specificcytokine pattern.

"Depletion", as used herein, can refer to an effective reduction in thenumber of cells belonging to a TH cell subpopulation, via, for example,a reduction in the proliferation of such TH cell subpopulation cells.The term can also refer to a decrease in the activity of cells belongingto a TH cell subpopulation, as would be evidenced, for example, by a percell decrease in the expression of the TH cell subpopulation-specificcytokine pattern.

The invention is based, in part on systematic search strategiesinvolving paradigms which utilize TH0, TH1, TH2, TH1-like and TH2-likecells, in systems which mimic the activity of the immune system orimmune disorders, coupled with sensitive and high-throughput geneexpression assays, to identify genes differentially expressed withinand/or among TH cell subpopulations. In contrast to approaches thatmerely evaluate the expression of a single known gene product presumedto play a role in some immune cell-related process or disorder, thesearch strategies and assays used herein permit the identification ofall genes, whether known or novel, which are differentially expressedwithin and among TH cell subpopulations, as well as making possible thecharacterization of their temporal regulation and function in the THcell response and/or in TH cell mediated disorders. This comprehensiveapproach and evaluation permits the discovery of novel genes and geneproducts, as well as the identification of a constellation of genes andgene products (whether novel or known) involved in novel pathways (e.g.,modulation pathways) that play a major role in the TH-cell mediatedimmune responses and TH cell subpopulation-related disorders. Thus, thepresent invention makes possible the identification and characterizationof targets useful for prognosis, diagnosis, monitoring, rational drugdesign, and/or therapeutic intervention of immune system disorders.

The Examples described in Sections 6 through 8, below, demonstrate thesuccessful use of the search strategies of the invention to identifygenes which are differentially expressed among and/or within TH cellsubpopulations.

The 102 and 103 genes represent genes which, while previously known, areshown here to be differentially expressed among TH cell subpopulations.Specifically, the 102 gene corresponds to the Granzyme A, or Hanukahfactor, gene, which encodes a trypsin-like serine protease. While thisgene had previously been reported to be expressed in natural killercells and a fraction of CD4⁺ cells, the results described herein reveal,for the first time, that the gene is differentially expressed within theTH2 cell subpopulation. Specifically, the 102 gene is expressed at alevel many-fold higher in the TH2 cell subpopulation than in the TH1cell subpopulation.

The 103 gene corresponds to a gene known as the T1, ST-2 or Fit-1 gene,which encodes, possibly via alternative splicing, both transmembrane andsoluble gene products. The gene 103 products belong to theimmunoglobulin superfamily, and bear a high resemblance to theinterleukin-1 (IL-1) receptor. The results presented herein demonstrate,for the first time, that this gene is expressed, in vivo, in a tightlycontrolled TH2-specific fashion. Thus, given its status as both a TH2cell subpopulation-specific marker and a cell surface protein, the gene103 products can be utilized in a variety of methods to diagnose and/ormodulate immune system disorders, in particular TH2 cellsubpopulation-related disorders.

In addition to these known genes, the systematic search strategiesdescribed herein were used to identify several novel genes which aredifferentially expressed within and/or among TH cell subpopulations.Specifically, these include the 10, 54, 57, 105, 106, 161 and 200 genes.

The 54, 105, 106 and 200 genes are each shown to be differentiallyexpressed within the TH1 cell subpopulation. specifically, these genesare expressed at levels many-fold higher in TH1 cell subpopulations thanin TH2 cell subpopulations.

The 10 and 57 genes represent TH inducible gene sequences. That is, theexpression of such genes in unstimulated TH cells is either undetectableor barely detectable, but is significantly upregulated in bothstimulated TH1 and stimulated TH2 cells. Thus, the 10 and 57 genesand/or their gene products can represent new targets for therapeutictreatment as part of a non-TH cell subpopulation dependent interventionprogram.

The 10 gene product is a 338 amino acid receptor molecule which is aparticularly suitable target for such a program in that the 10 geneproduct belongs to a class of proteins having a seven transmembranedomain sequence motif, which tend to represent G protein-coupledreceptor molecules. The 10 gene product structure, therefore, indicatesthat it may be involved in signal transduction events which may beimportant to T cell responses in general, and further indicates thatmodulation of 10 gene product may effectively ameliorate a wide range ofT cell-related disorders.

Specifically, because the 10 gene product is a transmembrane product,its activity, via either a physical change in the number of 10gene-expressing cells or by a change in the functional level of 10 geneproduct activity, can be particularly amenable to modulation. Forexample, natural ligands, derivatives of natural ligands and antibodieswhich bind to the 10 gene product can be utilized to reduce the numberof induced T cells present by either physically separating such cellsaway from other cells in a population, or, alternatively, by targetingthe specific destruction of the induced T cells or inhibiting theproliferation of such T cells.

Additionally, compounds such as 10 gene sequences or gene products suchas, for example, soluble 10 gene products, can be utilized to reduce thelevel of induced T cell activity, and, ultimately, bring about theamelioration of a wide range of T cell-related disorders. For example,the compounds can compete with the endogenous (i.e., natural) ligand forthe 10 gene product, leading to a modulation of induced T cell activity.Soluble proteins or peptides, such as peptides comprising one or more ofthe extracellular domains, or portions and/or analogs thereof, of the 10gene product, including, for example, soluble fusion proteins such asIg-tailed fusion proteins, can be particularly useful for this purpose.Additionally, antibodies directed against one or more of theextracellular portions of the 10 gene product may either reduce 10 geneproduct function by, for example, blocking ligand binding. Additionally,antibodies directed against the 10 gene product can, in certaininstances, serve to increase the level of 10 gene product activity.

The receptor nature of the 10 gene product makes possible useful methodsfor the identification of compounds which modulate the receptor'sfunctional activity and which can act as therapeutic agents in theamelioration of a wide range of T cell-related disorders. For example,functional assays which measure intracellular calcium release levels maybe utilized to identify compounds which act as either agonists orantagonists of 10 gene product activity. Such assays may, additionally,be utilized to identify the natural 10 gene product ligand. Stillfurther, any of these modulatory compounds can be utilized astherapeutic agents for the amelioration of a wide range of Tcell-related disorders.

Finally, the 161 gene is shown to be an additional new and potentiallyinteresting target for a therapeutic method aimed at the amelioration ofimmune disorder related symptoms. In fact, it is possible that 161 geneexpression may be indicative of the presence of yet another TH cellsubpopulation, in addition to TH1, TH2 and TH0 cell subpopulations.

The identification of TH cell subpopulation specific markers can beutilized in the treatment of a number of immune disorders, especially THcell subpopulation-related disorders. For example, markers for the TH2subpopulation can be used to ameliorate conditions involving aninappropriate IgE immune response, including but not limited to thesymptoms which accompany atopic conditions such as allergy and/orasthma. IgE-type antibodies are produced by stimulated B cells whichrequire, at least in part, IL-4 produced by the TH2 cell subpopulation.Therefore, a treatment which reduces the effective concentration ofsecreted IL-4, e.g., by reducing the activity or number of TH2 cells,will bring about a reduction in the level of circulating IgE, leading,in turn, to the amelioration or elimination of atopic conditions. Any ofthe TH2-specific gene products described herein can, therefore, be usedas a target to reduce or deplete the number and/or activity of TH2 cellsubpopulation cells for the treatment of such conditions.

The 103 gene can be particularly suitable for this purpose since one ofits gene products is a membrane-bound TH2 cell subpopulation molecule.Accordingly, natural ligands, derivatives of natural ligands andantibodies which bind to this 103 gene product, can be utilized toreduce the number of TH2 cells present by either physically separatingsuch cells away from other cells in a population, or, alternatively, bytargeting the specific destruction of TH2 cells or inhibiting theproliferation of such TH2 cells. Additionally, compounds such as 103gene sequences or gene products can be utilized to reduce the level ofTH2 cell activity, cause a reduction in IL-4 production, and,ultimately, bring about the amelioration of IgE related disorders. Forexample, the compounds can compete with the endogenous (i.e., natural)ligand for the 103 gene product. The resulting reduction in the amountof ligand-bound 103 gene transmembrane protein will modulate TH2cellular activity. Soluble proteins or peptides, such as peptidescomprising the extracellular domain, or portions and/or analogs thereof,of the 103 gene product, including, for example, soluble fusion proteinssuch as Ig-tailed fusion proteins, can be particularly useful for thispurpose.

The identification of TH cell subpopulation specific markers canadditionally be utilized in the treatment of a TH1 cellsubpopulation-related disorders. For example, markers for the TH1 cellsubpopulation can be used to ameliorate conditions involving aninappropriate cell-mediated immune response, including, but not limitedto chronic inflammatory and autoimmune disorders.

TH1 cell subpopulation specific gene sequences and/or gene products suchas the 54 (which encodes a 371 amino acid cysteine protease geneproduct), 105, 106 and 200 (which encodes a 280 amino acid transmembranegene product which is a member of the Ig superfamily) genes can,therefore, be suitable for ameliorating such TH1 cellsubpopulation-related disorders. The 200 gene product can beparticularly suitable for such a purpose in that it is not only TH1 cellsubpopulation-restricted, but the Ig superfamily 200 gene product is,additionally, membrane-bound. Therefore, natural ligands, derivatives ofnatural ligands and antibodies which bind to the 200 gene product can beutilized to reduce the number of TH1 cells present by either physicallyseparating such cells away from other cells in a population, or,alternatively, by targeting the specific destruction of TH1 cells orinhibiting the proliferation of such TH1 cells. Additionally, compoundssuch as 200 gene sequences or gene products such as soluble 200 geneproducts, can be utilized to reduce the level of TH2 cell activity, thusbringing about the amelioration of TH1 cell subpopulation-relateddisorders. For example, the compounds can compete with the endogenous(i.e., natural) ligand for the 200 gene product. The resulting reductionin the amount of ligand-bound 200 gene transmembrane protein willmodulate TH2 cellular activity. Soluble proteins or peptides, such aspeptides comprising the extracellular domain, or portions (such as, forexample, the Ig portion) and/or analogs thereof, of the 200 geneproduct, including, for example, soluble fusion proteins such asIg-tailed fusion proteins, can be particularly useful for this purpose.

3.1 DEFINITIONS

The term "TH cell subpopulation", as used herein, refers to a populationof TH cells exhibiting a gene expression pattern (e.g., a discretepattern of cytokines and/or receptor or other cell surface molecules)and activity which are distinct from the expression pattern and activityof other TH cells. Such TH cell subpopulations can include, but are notlimited to, TH0, TH1 and TH2 subpopulations, which will, for clarity andexample, and not by way of limitation, be frequently used herein asrepresentative TH cell subpopulations.

The term "TH-like cell subpopulation" (e.g., "TH1-like" or "TH2-like"),as used herein is intended to refer not only to a population of CD4⁺ THcells having the properties described, above, for a TH cellsubpopulation, but also refers to CD4⁻ cells, including CD8⁺ CTLs, whichexhibit TH-like cytokine expression patterns.

"Differential expression", as used herein, refers to both quantitativeas well as qualitative differences in the genes' temporal and/orcellular expression patterns.

"Target gene", as used herein, refers to a differentially expressed geneinvolved in immune disorders and/or in the differentiation, maintenanceand/or effector function of TH cell subpopulations, such that modulationof the level of target gene expression or of target gene productpresence and/or activity can, for example, act to result in the specificdepletion or repression, or, alternatively, the stimulation oraugmentation of one or more TH cell subpopulation, bringing about, inturn, the amelioration of symptoms of immune disorders, e.g., TH cellsubpopulation-related disorders. A target gene can also exhibitfingerprint and/or pathway gene characteristics.

"Fingerprint gene," as used herein, refers to a differentially expressedgene whose mRNA expression pattern, protein level and/or activity can beutilized as part of a prognostic or diagnostic in the evaluation ofimmune disorders, e.g., TH cell subpopulation-related disorders, orwhich, alternatively, can be used in methods for identifying compoundsuseful for the treatment of such disorders, by, for example, evaluatingthe effect of the compound on the fingerprint gene expression normallydisplayed in connection with the disease. A fingerprint gene can alsoexhibit target and/or pathway gene characteristics.

"Fingerprint pattern," as used herein, refers to the pattern generatedwhen the mRNA expression pattern, protein level and/or activity of aseries (which can range from two up to all the fingerprint genes whichexist for a given state) of fingerprint genes is determined. Afingerprint pattern can be a part of the same methods described, above,for the expression of a single fingerprint gene.

"Pathway genes", as used herein, refers to a gene whose product exhibitsan ability to interact with gene products involved in immune disorders,e.g., TH cell subpopulation-related disorders and/or to interact withgene products which are involved in the differentiation and effectorfunction of TH cell subpopulations. Pathway genes can also exhibittarget gene and/or fingerprint gene characteristics.

"Negative modulation", as used herein, refers to a reduction in thelevel and/or activity of target gene product relative to the leveland/or activity of the target gene product in the absence of themodulatory treatment. Alternatively, the term, as used herein, refers toa reduction in the number and/or activity of cells belonging to the THcell subpopulation relative to the number and/or activity of the TH cellsubpopulation in the absence of the modulatory treatment.

"Positive modulation", as used herein, refers to an increase in thelevel and/or activity of target gene product relative to the leveland/or activity of the gene product in the absence of the modulatorytreatment. Alternatively, the term, as used herein, refers to anincrease in the number and/or activity of cells belonging to the TH cellsubpopulation, relative to the number and/or activity of the TH cellsubpopulation in the absence of the modulatory treatment.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Differential display analysis of RNA from murine TH cellsubsets. Splenic T cells derived from T cell receptor transgenic micewere differentiated in vitro to become polarized populations of TH1 orTH2 subtypes. Lane 1: TH2 population 24 hours after tertiarystimulation; lane 2: TH1 population 24 hours after tertiary stimulation;lane 3: TH2 population 1 week after secondary stimulation; lane 4: TH1population 1 week after secondary stimulation; lane 5: TA3 cell line,which was used as antigen presenting cell (APC) for in vitrostimulation. (This sample was used as a negative control.) Each set oflanes consists of duplicates (a and b), in which cDNAs wereindependently generated from the same source of RNA. Arrow points todifferentially expressed sequence, which is referred to herein as band102.

Further, the gene corresponding to band 102 is referred to herein as the102 gene. All lanes are products of a polymerase chain reaction (PCR) inwhich T₁₁ GG was used as the 3' oligonucleotide and a random 10meroligonucleotide (Oligo #4, OP-D kit, Operon, Inc.) was used as the 5'oligonucleotide.

FIG. 2. Nucleotide sequence of clone 102.1 of band 102 (SEQ. ID NO: 1).The gene corresponding to band 102 is referred to herein as the 102gene.

FIG. 3. Northern blot analysis of confirming differential regulation ofthe 102 gene within primary TH1/TH2 cultures and murine tissues. RNA washarvested from T cell lines derived from a T cell receptor transgenicstrain stimulated in vitro. Lane 1, TH2, 40 hours after secondstimulation; lane 2, TH1, 40 hours after second stimulation; lane 3, TH2population 24 hours after tertiary stimulation; lane 4, TH1, 24 hoursafter tertiary stimulation; lane 5, murine thymus; lane 6, murinespleen. Five micrograms of total RNA was used per lane. The cloned band102 sequence was used as a probe.

FIG. 4A. Nucleotide sequence clone 103.1 of band 103 (SEQ ID NO:2). Thegene corresponding to band 103 is referred to herein as gene 103.

FIG. 4B. 103 gene products. This diagram illustrates the relationshipbetween band 103, 103 gene (also known as ST-2, T1 and Fit-1) productsand the IL-1 receptor polypeptide structure. The extracellular,transmembrane and cytoplasmic domains of the proteins are noted, alongwith the amino acid residues marking the boundaries of these domains.(Adapted from Yanagisawa et al., 1993, FEBS Lett. 318:83-87.)

FIG. 5. Quantitative RT-PCR analysis of 103 gene expression in polarizedpopulations of murine TH cells. RNA samples were harvested from culturedT cell populations 24 hours after tertiary stimulation with antigen.cDNA samples were PCR amplified and the products of those reactions wereelectrophoresed on a 1% agarose gel and visualized by ethidium bromidestaining. 103 gene expression is shown in the upper panel. γ-actin data,bottom panel, was included as a control for differences in samplequality. The numbers above each lane represent the dilution factors ofeach cDNA. The same cDNA samples were used for both the 103 gene and theγ-actin amplifications.

FIG. 6. Northern blot analysis of 103 gene expression in representativemurine TH cell lines (TH2: CDC25, D10.G4, DAX; TH1: AE7.A, Dorris,D1.1). Clones were either unstimulated (-) or stimulated (+) for 6 hourswith plate-bound anti-CD3 antibody. Ten micrograms of total RNA wereloaded per lane. The positions of 18s and 28s ribosomal RNA are shown asreference markers.

FIG. 7. Northern blot analysis of 103 gene expression in T cell clonesand murine tissues. Lane 1: DAX cells, no stimulation; lane 2, AE7cells, stimulation; lane 3, AE7 cells, no stimulation; lane 4, D10.G4cells, stimulation; lane 5, D10.G4 cells, no stimulation; lane 6, brain;lane 7, heart; lane 8, lung; lane 9, spleen; lane 10, liver. Clones werestimulated with plate-bound anti-CD3 antibody for 6 hours. 7.5 and 10micrograms total RNA was used for each cell line and each tissue,respectively. a, b, and c arrows refer to RNA encoding full length (a)and truncated (b,c) forms of the 103 gene. The positions of 18s and 28sribosomal RNA markers are shown.

FIG. 8. RNAse protection analysis of 103 gene mRNA, illustratingregulation of 103 gene expression in murine TH cell clones. Lanes 2-6:β-actin protection; lanes 9-13: 103 gene protection; lanes 1 and 8:markers; lanes 2 and 9: unstimulated TH1 clones; lanes 3 and 10:stimulated TH1 clones; lanes 4 and 11: unstimulated TH2 clones; lanes 5and 12: stimulated TH2 clones; lanes 6 and 13: fully RNAse A digestedunprotected probe; lanes 7 and 14: probe alone, in absence of addedRNAse.

Expected Fragment Sizes

β-actin protected probe: 250 nucleotides;

β-actin full length probe: 330 nucleotides;

103 gene long form fragment: 257 nucleotides;

103 gene short form fragment: 173 nucleotides;

103 gene full length probe: 329 nucleotides.

FIG. 9. The full length 10 gene nucleotide sequence (SEQ ID NO: 3) isshown on the top line, while the derived amino acid sequence of the 10gene product (SEQ ID NO: 9) is shown on the bottom line. The underlinedportion of the nucleotide sequence corresponds to the band 10 nucleotidesequence. The data shown in FIG. 10A-C was obtained through the use ofthe portion of the 10 gene product which is encoded by the band 10nucleotide sequence.

FIG. 10A-C. 10 gene hydrophilicity data, indicating that the 10gene-derived amino acid sequence predicts the presence of a seventransmembrane domain structural motif. 10A) platelet activating factorreceptor hydrophilicity plot illustrating the protein's seventransmembrane domain structural motif; 10B) 10 gene hydrophilicity plotillustrating a portion of the protein's putative seven transmembranedomain structural motif; 10C) platelet activating factor receptorhydrophilicity plot illustrating part of the protein's seventransmembrane structural motif.

FIG. 11. Chromosomal mapping of locus containing the 10 gene sequence. Amap of a portion of mouse chromosome 12 is shown. Numbers to left ofchromosome are in centiMorgans; D12NDS11, D12MIT4, and D12MIT8 representmouse microsatellite markers; TH10 represents 10 gene.

FIG. 12. Nucleotide sequence of clone 7 of band 57 (SEQ ID NO:4). Thegene corresponding to band 57 is referred to herein as the 57 gene.

FIG. 13. Consensus nucleotide sequence of band 105 (SEQ ID NO:5). "N"signifies "any nucleotide". The gene corresponding to band 105 isreferred to herein as the 105 gene.

FIG. 14. Nucleotide sequence obtained from clone H of band 106 (SEQ IDNO:6). "N" signifies "any nucleotide". The gene corresponding to band106 is referred to herein as the 106 gene.

FIG. 15. Nucleotide sequence of clone G of band 161 (SEQ ID NO:7). Thegene corresponding to band 161 is referred to herein as the 161 gene.

FIG. 16. Multiple sequence alignment of 161 clone G SEQ ID NO:24 withamino acid sequences identified in a BLAST search [CHIMP GENE: (SEQ IDNO:13); HUMAN 6-16: (SEQ ID NO:14); HUMAN 6-16: (SEQ ID NO:15); HUMAN6-16: (SEQ ID NO:16); HUMAN P27: (SEQ ID NO:17)]. Asterisks signifypositions that are identical; dots indicate conserved positions.

FIG. 17. Nucleotide and amino acid sequence of the full length 200 gene.Bottom line: 200 gene nucleotide sequence (SEQ ID NO:8); top line: 200gene product derived amino acid sequence (SEQ ID NO: 10).

FIG. 18. Northern blot analysis of 200 gene expression in representativemurine TH cell lines (TH2: CDC25, D10.G4, DAX; TH1: AE7.A, Dorris,D1.1). Clones were either unstimulated (-) or stimulated (+) for 6 hourswith plate-bound anti-CD3 antibody. The positions of RNA markers, inkilobases, are shown for reference. The arrow marks the position of 200gene mRNA.

FIG. 19. Northern blot analysis of 54 gene expression within TH1 (D1.1,Dorris, AE7) cell lines and TH2 (D10.G4, DAX, CDC25) cell lines, eitherstimulated (+) or unstimulated (-) with anti-CD3 antibodies. 15micrograms of total RNA were loaded per lane. Cells were stimulatedbetween 6 and 7 hours with anti-CD3 antibodies, as described, below, inSection 8.1. The Northern blots were hybridized with a probe made fromthe entire band 54 nucleotide sequence.

FIG. 20. Northern blot analysis of gene 54 time course study. RNA fromTH1 cell line AE7 cells was isolated, either unstimulated or stimulatedfor varying periods of time, as indicated. Second, RNA from two TH2 celllines (DAX, CDC25) was isolated from either unstimulated cells or fromcells which had been stimulated for two hours with anti-CD3 antibodies.15 micrograms total RNA were loaded per lane. A band 54 DNA probe wasused for hybridization.

FIG. 21. Northern blot analysis of 54 gene expression in varioustissues. 15 micrograms of total RNA were loaded per lane. A band 54 DNAprobe was used for hybridization.

FIG. 22. Nucleotide and amino acid sequence of the full length 54 gene.Bottom line: 54 gene nucleotide sequence (SEQ ID NO:11). Top line: 54gene derived amino acid sequence (SEQ ID NO:12).

FIG. 23. The 54 gene product bears a high level of homology to thecysteine protease class of proteins. The 54 gene product amino acid isdepicted with its predicted prepro sequence and mature cysteine proteasepolypeptide sequence identified. The individual boxed amino acidresidues represent residues thought to lie within the cysteine proteaseactive site and the stretch of amino acid residues which are boxedrepresent a region with homology to a stretch of amino acid residuesnormally seen within the preproenzyme portion of cysteine proteasemolecules. The circled amino acid residues within this stretch representconserved amino acids. The arrow indicates the putativepost-translational cleavage site.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the treatment and diagnosis of immunedisorders, especially TH cell subpopulation-related disorders,including, but not limited to, atopic conditions, such as asthma andallergy, including allergic rhinitis, psoriasis, the effects of pathogeninfection, chronic inflammatory diseases, organ-specific autoimmunity,graft rejection and graft versus host disease, are described. Theinvention is based, in part, on the evaluation of the expression androle of all genes that are differentially expressed within and/or amongTH cell subpopulations in paradigms that are physiologically relevant toTH-mediated immune response and/or TH-subpopulation related disorders.This permits the definition of disease pathways that are useful bothdiagnostically and therapeutically.

Genes, termed "target genes" and/or "fingerprint genes", which aredifferentially expressed within and among TH cells and TH cellsubpopulations in normal and/or disease states, and/or during thedifferentiation into such mature subpopulations are described in Section5.4. Additionally, genes, termed "pathway genes", whose gene productsexhibit an ability to interact with gene products involved in TH cellsubpopulation-related disorders and/or with gene products which areinvolved in the differentiation and effector function of thesubpopulations are described in Section 5.4. Pathway genes canadditionally have fingerprint and/or target gene characteristics.Methods for the identification of such fingerprint, target, and pathwaygenes are also described in Sections 5.1 and 5.2.

Further, the gene products of such fingerprint, target, and pathwaygenes are described in Section 5.5, antibodies to such gene products aredescribed in Section 5.6, as are cell- and animal-based models of THcell subpopulation differentiation and TH cell subpopulation-relateddisorders to which such gene products can contribute in Section 5.7.

Methods for prognostic and diagnostic evaluation of various TH cellsubpopulation-related disorders, for the identification of subjectsexhibiting a predisposition to such disorders, and for monitoring theefficacy of compounds used in clinical trials are described in Section5.11.

Methods for the identification of compounds which modulate theexpression of genes or the activity of gene products involved in TH cellsubpopulation-related disorders and to the differentiation and effectorfunction of TH cell subpopulations are described in Section 5.8, andmethods for the treatment of immune disorders are described in Section5.9.

5.1 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

Described herein are methods for the identification of differentiallyexpressed genes which are involved in immune disorders, e.g., TH cellsubpopulation-related disorders, and/or which are involved in thedifferentiation, maintenance and effector function of thesubpopulations. There exist a number of levels at which the differentialexpression of such genes can be exhibited. For example, differentialexpression can occur in undifferentiated TH cells versus differentiatedor differentiating TH cells (although not necessarily within one TH cellsubpopulation versus another), in naive TH cells versus memory TH cells,within one TH cell subpopulation versus another (e.g., TH1 versus TH2subpopulations), in mature, stimulated cells versus mature, unstimulatedcells of a given TH cell subpopulation or in TH cellsubpopulation-related disorder states relative to their expression innormal, or non-TH cell subpopulation-related disorder states. Suchdifferentially expressed genes can represent target and/or fingerprintgenes.

Methods for the identification of such differentially expressed genesare described, below, in Section 5.1.1. Methods for the furthercharacterization of such differentially expressed genes, and for theircategorization as target and/or fingerprint genes, are presented, below,in Section 5.3.

"Differential expression" as used herein refers to both quantitative aswell as qualitative differences in the genes' temporal and/or cell typeexpression patterns. Thus, a differentially expressed gene canqualitatively have its expression activated or completely inactivatedin, for example, normal versus TH cell subpopulation-related disorderstates, in one TH cell subpopulation versus another (e.g., TH1 versusTH2), in antigen stimulated versus unstimulated sets of TH cells, or inundifferentiated versus differentiated or differentiating TH cells. Sucha qualitatively regulated gene will exhibit an expression pattern withina state or cell type which is detectable by standard techniques in onesuch state or cell type, but is not detectable in both.

Alternatively, a differentially expressed gene can exhibit an expressionlevel which differs, i.e., is quantitatively increased or decreased, innormal versus TH cell subpopulation-related disorder states, in antigenstimulated versus unstimulated sets of TH cells, in one TH cellsubpopulation versus another, or in undifferentiated versusdifferentiated or differentiating TH cells. Because differentiation is amultistage event, genes which are differentially expressed can also beidentified at any such intermediate differentiative stage.

The degree to which expression differs need only be large enough to bevisualized via standard characterization techniques, such as, forexample, the differential display technique described below. Other suchstandard characterization techniques by which expression differences canbe visualized include, but are not limited to, quantitative RT (reversetranscriptase) PCR and Northern analyses and RNase protectiontechniques.

Differentially expressed genes can be further described as target genesand/or fingerprint genes. "Fingerprint gene," as used herein, refers toa differentially expressed gene whose expression pattern can be utilizedas part of a prognostic or diagnostic TH cell subpopulation-relateddisorder evaluation, or which, alternatively, can be used in methods foridentifying compounds useful for the treatment of TH cellsubpopulation-related disorders. A fingerprint gene can also have thecharacteristics of a target gene or a pathway gene (see below, inSection 5.2).

"Fingerprint pattern," as used herein, refers to the pattern generatedwhen the expression pattern of a series (which can range from two up toall the fingerprint genes which exist for a given state) of fingerprintgenes is determined. A fingerprint pattern can also be used in methodsfor identifying compounds useful in the treatment of immune disorders,e.g., by evaluating the effect of the compound on the fingerprintpattern normally displayed in connection with the disease.

"Target gene", as used herein, refers to a differentially expressed geneinvolved in TH cell subpopulation-related disorders and/or indifferentiation, maintenance and/or effector function of thesubpopulations in a manner by which modulation of the level of targetgene expression or of target gene product activity can act to amelioratesymptoms of TH cell subpopulation-related disorders. For example, suchmodulation can result either the depletion or stimulation of one or moreTH cell subpopulation, which, in turn, brings about the amelioration ofimmune disorder, e.g., TH cell subpopulation disorder, symptoms.

"Stimulation", as used herein, can refer to an effective increase in thenumber of cells belonging to a T cell population, such as a TH cellsubpopulation, via, for example, the proliferation of such TH cellsubpopulation cells. The term can also refer to an increase in theactivity of cells belonging to a TH cell subpopulation, as would byevidenced, for example, by a per cell increase in the expression of theTH cell subpopulation-specific cytokine pattern.

"Depletion", as used herein, can refer to an effective reduction in thenumber of cells belonging to a T cell population, such as a TH cellsubpopulation, via, for example, a reduction in the proliferation ofsuch TH cell subpopulation cells. The term can also refer to a decreasein the activity of cells belonging to a TH cell subpopulation, as wouldbe evidenced, for example, by a per cell decrease in the expression ofthe TH cell subpopulation-specific cytokine pattern.

TH cell subpopulation-related disorders include, for example, atopicconditions, such as asthma and allergy, including allergic rhinitis, theeffects of pathogen, including viral, infection, chronic inflammatorydiseases, psoriasis, glomerular nephritis, organ-specific autoimmunity,graft rejection and graft versus host disease. A target gene can alsohave the characteristics of a fingerprint gene and/or a pathway gene (asdescribed, below, in Section 5.2).

5.1.1 METHODS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

A variety of methods can be utilized for the identification of geneswhich are involved in immune disorder states, e.g., TH cellsubpopulation-related disorder states, and/or which are involved indifferentiation, maintenance and/or effector function of thesubpopulations. Described in Section 5.1.1.1 are experimental paradigmswhich can be utilized for the generation of subjects and samples whichcan be used for the identification of such genes. Material generated inparadigm categories can be characterized for the presence ofdifferentially expressed gene sequences as discussed, below, in Section5.1.1.2.

5.1.1.1 PARADIGMS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSEDGENES

Paradigms which represent models of normal and abnormal immune responsesare described herein. These paradigms can be utilized for theidentification of genes which are differentially expressed within andamong TH cell subpopulations, including but not limited to TH1 and TH2subpopulations. Such genes can be involved in, for example, TH cellsubpopulation differentiation, maintenance, and/or effector function,and in TH cell subpopulation-related disorders. For example, TH cellscan be induced to differentiate into either TH1 or TH2 states, can bestimulated with, for example, a foreign antigen, and can be collected atvarious points during the procedure for analysis of differential geneexpression.

In one embodiment of such a paradigm, referred to herein as the"transgenic T cell paradigm", transgenic animals, preferably mice, areutilized which have been engineered to express a particular T cellreceptor, such that the predominant T cell population of the immunesystem of such a transgenic animal recognizes only one antigen. Such asystem is preferred in that it provides a source for a large populationof identical T cells whose naivete can be assured, and whose response tothe single antigen it recognizes is also assured. T helper cellsisolated from such a transgenic animal are induced, in vitro, todifferentiate into TH cell subpopulations such as TH1, TH2, or TH0 cellsubpopulations. In a specific embodiment, one T helper cell group (theTH1 group) is exposed to IL-12, a cytokine known to inducedifferentiation into the TH1 state, a second T helper cell group (theTH2 group) is exposed to IL-4, a cytokine known to inducedifferentiation into the TH2 state, and a third group is allowed, by alack of cytokine-mediated induction, to enter a TH-undirected state.

A second paradigm, referred to herein as a "cell line paradigm", can beutilized which uses mature TH cell clones, such as TH1 and TH2 andTH1-like and TH2-like cell lines, preferably human cell lines. Such THcell lines can include, but are not limited to the following well knownmurine cell lines: Doris, AE7, D10.G4, DAX, D1.1 and CDC25. Such T celllines can be derived from normal individuals as well as individualsexhibiting TH cell subpopulation-related disorders, such as, forexample, chronic inflammatory diseases and disorders, such as Crohn'sdisease, reactive arthritis, including Lyme disease, insulin-dependentdiabetes, organ-specific autoimmunity, including multiple sclerosis,Hashimoto's thyroiditis and Grave's disease, contact dermatitis,psoriasis, graft rejection, graft versus host disease, sarcoidosis,atopic conditions, such as asthma and allergy, including allergicrhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis) and certainviral infections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

The TH cell clones can be stimulated in a variety of ways. Suchstimulation methods include, but are not limited to, pharmacologicalmethods, such as exposure to phorbol esters, calcium ionophores, orlectins (e.g., Concanavalin A), by treatment with antibodies directedagainst T-cell receptor epitopes (e.g., anti-CD3 antibodies) orexposure, in the presence of an appropriate antigen presenting cell(APC), to an antigen that the particular TH cells are known torecognize. Following such primary stimulation, the cells can bemaintained in culture without stimulation and, for example, in thepresence of IL-2, utilizing standard techniques well known to those ofskill in the art. The cells can then be exposed to one or moreadditional cycles of stimulation and maintenance.

A third paradigm, referred to herein as an "in vivo paradigm", can alsobe utilized to discover differentially expressed gene sequences. In vivostimulation of animal models forms the basis for this paradigm. The invivo nature of the stimulation can prove to be especially predictive ofthe analogous responses in living patients. Stimulation can beaccomplished via a variety of methods. For example, animals, such astransgenic animals described earlier in this Section, can be injectedwith appropriate antigen and appropriate cytokine to drive the desiredTH cell differentiation. Draining lymph nodes can then be harvested atvarious time points after stimulation. Lymph nodes from, for example,TH1-directed animals can be compared to those of TH2-directed animals.

A wide range of animal models, representing both models of normal immunedifferentiation and function as well as those representing immunedisorders can be utilized for this in vivo paradigm. For example, any ofthe animal models, both recombinant and non-recombinant, described,below, in Section 5.7.1, can be used.

Cell samples can be collected during any point of such a procedure. Forexample, cells can be obtained following any stimulation period and/orany maintenance period. Additionally, cells can be collected duringvarious points during the TH cell differentiation process. RNA collectedfrom such samples can be compared and analyzed according to, forexample, methods described, below, in Section 5.1.1.2. For example, RNAfrom TH0, TH1 and TH2 groups isolated at a given time point can then beanalyzed and compared. Additionally, RNA from stimulated andnon-stimulated cells within a given TH cell group can also be comparedand analyzed. Further, RNA collected from undifferentiated TH cells canbe compared to RNA collected from cells at various stages during thedifferentiative process which ultimately yields TH cell subpopulations.

5.1.1.2 ANALYSIS OF PARADIGM MATERIAL

In order to identify differentially expressed genes, RNA, either totalor mRNA, can be isolated from the TH cells utilized in paradigms such asthose described in Section 5.1.1.1. Any RNA isolation technique whichdoes not select against the isolation of mRNA can be utilized for thepurification of such RNA samples. See, for example, Ausubel, F. M. etal., eds., 1987-1993, Current Protocols in Molecular Biology, John Wiley& Sons, Inc. New York, which is incorporated herein by reference in itsentirety. Additionally, large numbers of cell samples can readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,P. (1989, U.S. Pat. No. 4,843,155), which is incorporated herein byreference in its entirety.

Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes can be identified byutilizing a variety of methods which are well known to those of skill inthe art. For example, differential screening (Tedder, T. F. et al.,1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization(Hedrick, S. M. et al., 1984, Nature 308:149-153; Lee, S. W. et al.,1984, Proc. Natl. Acad. Sci. USA 88:2825), and, preferably, differentialdisplay (Liang, P. and Pardee, A. B., 1992, Science 257:967-971; U.S.Pat. No. 5,262,311, which are incorporated herein by reference in theirentirety), can be utilized to identify nucleic acid sequences derivedfrom genes that are differentially expressed.

Differential screening involves the duplicate screening of a cDNAlibrary in which one copy of the library is screened with a total cellcDNA probe corresponding to the mRNA population of one cell type while aduplicate copy of the cDNA library is screened with a total cDNA probecorresponding to the mRNA population of a second cell type. For example,one cDNA probe can correspond to a total cell cDNA probe of a cell typeor tissue derived from a control subject, while the second cDNA probecan correspond to a total cell cDNA probe of the same cell type ortissue derived from an experimental subject. Those clones whichhybridize to one probe but not to the other potentially represent clonesderived from genes differentially expressed in the cell type of interestin control versus experimental subjects.

Subtractive hybridization techniques generally involve the isolation ofmRNA taken from two different sources, the hybridization of the mRNA orsingle-stranded cDNA reverse-transcribed from the isolated mRNA, and theremoval of all hybridized, and therefore double-stranded, sequences. Theremaining non-hybridized, single-stranded cDNAs, potentially representclones derived from genes that are differentially expressed among thetwo mRNA sources. Such single-stranded cDNAs are then used as thestarting material for the construction of a library comprising clonesderived from differentially expressed genes.

The differential display technique is a procedure, utilizing thewell-known polymerase chain reaction (PCR; the experimental embodimentset forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202), which allowsfor the identification of sequences derived from genes which aredifferentially expressed. First, isolated RNA is reverse-transcribedinto single-stranded cDNA, utilizing standard techniques which are wellknown to those of skill in the art. Primers for the reversetranscriptase reaction can include, but are not limited to, oligodT-containing primers, preferably of the 3' primer type ofoligonucleotide described below.

Next, this technique uses pairs of PCR primers, as described below,which allow for the amplification of clones representing a reproduciblesubset of the RNA transcripts present within any given cell. Utilizingdifferent pairs of primers allows each of the primed mRNA transcriptspresent in a cell to be amplified. Among such amplified transcripts canbe identified those which have been produced from differentiallyexpressed genes.

The 3' oligonucleotide primer of the primer pairs can contain an oligodT stretch of 10-13, preferably 11, dT nucleotides at its 5' end, whichhybridizes to the poly(A) tail of mRNA or to the complement of a cDNAreverse transcribed from an mRNA poly(A) tail. In order to increase thespecificity of the 3' primer, the primer can contain one or more,preferably two, additional nucleotides at its 3' end. Because,statistically, only a subset of the mRNA derived sequences present inthe sample of interest will hybridize to such primers, the additionalnucleotides allow the primers to amplify only a subset of the mRNAderived sequences present in the sample of interest. This is preferredin that it allows more accurate and complete visualization andcharacterization of each of the bands representing amplified sequences.

The 5' primer can contain a nucleotide sequence expected, statistically,to have the ability to hybridize to cDNA sequences derived from thecells or tissues of interest. The nucleotide sequence can be anarbitrary one, and the length of the 5' oligonucleotide primer can rangefrom about 9 to about 15 nucleotides, with about 13 nucleotides beingpreferred.

Arbitrary primer sequences cause the lengths of the amplified partialcDNAs produced to be variable, thus allowing different clones to beseparated by using standard denaturing sequencing gel electrophoresis.

PCR reaction conditions should be chosen which optimize amplifiedproduct yield and specificity, and, additionally, produce amplifiedproducts of lengths which can be resolved utilizing standard gelelectrophoresis techniques. Such reaction conditions are well known tothose of skill in the art, and important reaction parameters include,for example, length and nucleotide sequence of oligonucleotide primersas discussed above, and annealing and elongation step temperatures andreaction times.

The pattern of clones resulting from the reverse transcription andamplification of the mRNA of two different cell types is displayed viasequencing gel electrophoresis and compared. Differentially expressedgenes are indicated by differences in the two banding patterns.

Once potentially differentially expressed gene sequences have beenidentified via bulk techniques such as, for example, those describedabove, the differential expression of such putatively differentiallyexpressed genes should be corroborated. Corroboration can beaccomplished via, for example, such well known techniques as Northernanalysis, quantitative RT/PCR, or RNAse protection.

Upon corroboration, the differentially expressed genes can be furthercharacterized, and can be identified as target and/or fingerprint genes,as discussed, below, in Section 5.3.

The amplified sequences of differentially expressed genes obtainedthrough, for example, differential display can be used to isolate fulllength clones of the corresponding gene. The full length coding portionof the gene can readily be isolated, without undue experimentation, bymolecular biological techniques well known in the art. For example, theisolated differentially expressed amplified fragment can be labeled andused to screen a cDNA library. Alternatively, the labeled fragment canbe used to screen a genomic library.

PCR technology can also be utilized to isolate full length cDNAsequences. As described, above, in this Section, the isolated, amplifiedgene fragments obtained through differential display have 5' terminalends at some random point within the gene and usually have 3' terminalends at a position corresponding to the 3' end of the transcribedportion of the gene. Once nucleotide sequence information from anamplified fragment is obtained, the remainder of the gene (i.e., the 5'end of the gene, when utilizing differential display) can be obtainedusing, for example, RT-PCR.

In one embodiment of such a procedure for the identification and cloningof full length gene sequences, RNA can be isolated, following standardprocedures, from an appropriate tissue or cellular source. A reversetranscription reaction can then be performed on the RNA using anoligonucleotide primer complimentary to the mRNA that corresponds to theamplified fragment, for the priming of first strand synthesis. Becausethe primer is anti-parallel to the mRNA, extension will proceed towardthe 5' end of the mRNA. The resulting RNA/DNA hybrid can then be"tailed" with guanines using a standard terminal transferase reaction,the hybrid can be digested with RNAase H, and second strand synthesiscan then be primed with a poly-C primer. Using the two primers, the 5'portion of the gene is amplified using PCR. Sequences obtained can thenbe isolated and recombined with previously isolated sequences togenerate a full-length cDNA of the differentially expressed genes of theinvention. For a review of cloning strategies and recombinant DNAtechniques, see e.g., Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, (Volumes 1-3) Cold Spring Harbor Press, N.Y.; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

5.2 METHODS FOR THE IDENTIFICATION OF PATHWAY GENES

Methods are described herein for the identification of pathway genes."Pathway gene", as used herein, refers to a gene whose gene productexhibits the ability to interact with gene products involved in TH cellsubpopulation-related disorders and/or to interact with gene productswhich are involved in differentiation, maintenance and/or effectorfunction of TH cell subpopulations. A pathway gene can be differentiallyexpressed and, therefore, can have the characteristics of a targetand/or fingerprint gene, as described, above, in Section 5.1.

Any method suitable for detecting protein-protein interactions can beemployed for identifying pathway gene products by identifyinginteractions between gene products and gene products known to beinvolved in TH cell subpopulation-related disorders and/or involved indifferentiation, maintenance, and/or effector function of thesubpopulations. Such known gene products can be cellular orextracellular proteins. Those gene products which interact with suchknown gene products represent pathway gene products and the genes whichencode them represent pathway genes.

Among the traditional methods which can be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of pathway gene products. Once identified,a pathway gene product can be used, in conjunction with standardtechniques, to identify its corresponding pathway gene. For example, atleast a portion of the amino acid sequence of the pathway gene productcan be ascertained using techniques well known to those of skill in theart, such as via the Edman degradation technique (see, e.g., Creighton,1983, "Proteins: Structures and Molecular Principles", W. H. Freeman &Co., N.Y., pp.34-49). The amino acid sequence obtained can be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for pathway gene sequences. Screening can be accomplished, forexample, by standard hybridization or PCR techniques. Techniques for thegeneration of oligonucleotide mixtures and for screening are well-known.(See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods andApplications, 1990, Innis, M. et al., eds. Academic Press, Inc., NewYork).

Additionally, methods can be employed which result in the simultaneousidentification of pathway genes which encode proteins interacting with aprotein involved in TH cell subpopulation-related disorder states and/ordifferentiation, maintenance, and/or effector function of thesubpopulations. These methods include, for example, probing expressionlibraries with labeled protein known or suggested to be involved in thedisorders and/or the differentiation, maintenance, and/or effectorfunction of the subpopulations, using this protein in a manner similarto the well known technique of antibody probing of λgt11 libraries.

One method which detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration purposes only and not byway of limitation. One version of this system has been described (Chienet al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to a known protein, in this case,a protein known to be involved in TH cell subpopulation differentiationor effector function, or in TH cell subpopulation-related disorders, andthe other consists of the activator protein's activation domain fused toan unknown protein that is encoded by a cDNA which has been recombinedinto this plasmid as part of a cDNA library. The plasmids aretransformed into a strain of the yeast Saccharomyces cerevisiae thatcontains a reporter gene (e.g., lacZ) whose regulatory region containsthe transcription activator's binding sites. Either hybrid protein alonecannot activate transcription of the reporter gene, the DNA-bindingdomain hybrid cannot because it does not provide activation function,and the activation domain hybrid cannot because it cannot localize tothe activator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

The two-hybrid system or related methodology can be used to screenactivation domain libraries for proteins that interact with a known"bait" gene product. By way of example, and not by way of limitation,gene products known to be involved in TH cell subpopulation-relateddisorders and/or differentiation, maintenance, and/or effector functionof the subpopulations can be used as the bait gene products. Totalgenomic or cDNA sequences are fused to the DNA encoding an activationdomain. This library and a plasmid encoding a hybrid of the bait geneproduct fused to the DNA-binding domain are cotransformed into a yeastreporter strain, and the resulting transformants are screened for thosethat express the reporter gene. For example, and not by way oflimitation, the bait gene can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. These colonies are purified and the library plasmidsresponsible for reporter gene expression are isolated. DNA sequencing isthen used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait gene product are to be detected can be made using methods routinelypracticed in the art. According to the particular system describedherein, for example, the cDNA fragments can be inserted into a vectorsuch that they are translationally fused to the activation domain ofGAL4. This library can be co-transformed along with the bait gene-GAL4fusion plasmid into a yeast strain which contains a lacZ gene driven bya promoter which contains GAL4 activation sequence. A cDNA encodedprotein, fused to GAL4 activation domain, that interacts with bait geneproduct will reconstitute an active GAL4 protein and thereby driveexpression of the lacZ gene. Colonies which express lacZ can be detectedby their blue color in the presence of X-gal. The cDNA can then bepurified from these strains, and used to produce and isolate the baitgene-interacting protein using techniques routinely practiced in theart.

Once a pathway gene has been identified and isolated, it can be furthercharacterized as, for example, discussed below, in Section 5.3.

5.3 CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

Differentially expressed genes, such as those identified via the methodsdiscussed, above, in Section 5.1, and pathway genes, such as thoseidentified via the methods discussed, above, in Section 5.2, above, aswell as genes identified by alternative means, can be furthercharacterized by utilizing, for example, methods such as those discussedherein. Such genes will be referred to herein as "identified genes".

Analyses such as those described herein yield information regarding thebiological function of the identified genes. An assessment of thebiological function of the differentially expressed genes, in addition,will allow for their designation as target and/or fingerprint genes.

Specifically, any of the differentially expressed genes whose furthercharacterization indicates that a modulation of the gene's expression ora modulation of the gene product's activity can ameliorate any of the THcell subpopulation-related disorders of interest will be designated"target genes", as defined, above, in Section 5.1. Such target genes andtarget gene products, along with those discussed below, will constitutethe focus of the compound discovery strategies discussed, below, inSection 5.8. Further, such target genes, target gene products and/ormodulating compounds can be used as part of the TH cellsubpopulation-disorder treatment methods described, below, in Section5.9. Such methods can include, for example, methods whereby the TH cellsubpopulation of interest is selectively depleted or repressed, or,alternatively, stimulated or augmented.

Any of the differentially expressed genes whose further characterizationindicates that such modulations can not positively affect TH cellsubpopulation-related disorders of interest, but whose expressionpattern contributes to a gene expression "fingerprint" patterncorrelative of, for example, a TH1/TH2-related disorder state, will bedesignated a "fingerprint gene". "Fingerprint patterns" will be morefully discussed, below, in Section 5.11.1. It should be noted that eachof the target genes can also function as fingerprint genes, as well ascan all or a portion of the pathway genes.

It should further be noted that the pathway genes can also becharacterized according to techniques such as those described herein.Those pathway genes which yield information indicating that modulationof the gene's expression or a modulation of the gene product's activitycan ameliorate any a TH cell subpopulation-related disorder will be alsobe designated "target genes". Such target genes and target geneproducts, along with those discussed above, will constitute the focus ofthe compound discovery strategies discussed, below, in Section 5.8 andcan be used as part of the treatment methods described in Section 5.9,below.

In instances wherein a pathway gene's characterization indicates thatmodulation of gene expression or gene product activity can notpositively affect TH cell subpopulation-related disorders of interest,but whose expression is differentially expressed and contributes to agene expression fingerprint pattern correlative of, for example, aTH1/TH2-related disorder state, such pathway genes can additionally bedesignated as fingerprint genes.

A variety of techniques can be utilized to further characterize theidentified genes. First, the nucleotide sequence of the identifiedgenes, which can be obtained by utilizing standard techniques well knownto those of skill in the art, can, for example, be used to revealhomologies to one or more known sequence motifs which can yieldinformation regarding the biological function of the identified geneproduct.

Second, an analysis of the tissue and/or cell type distribution of themRNA produced by the identified genes can be conducted, utilizingstandard techniques well known to those of skill in the art. Suchtechniques can include, for example, Northern, RNAse protection, andRT-PCR analyses. Such analyses provide information as to, for example,whether the identified genes are expressed in cell types expected tocontribute to the specific TH cell subpopulation-related disorders ofinterest. Such analyses can also provide quantitative informationregarding steady state mRNA regulation, yielding data concerning whichof the identified genes exhibits a high level of regulation in celltypes which can be expected to contribute to the TH cellsubpopulation-related disorders of interest. Additionally, standard insitu hybridization techniques can be utilized to provide informationregarding which cells within a given tissue or population of cellsexpress the identified gene. Such an analysis can provide informationregarding the biological function of an identified gene relative to agiven TH cell subpopulation-related disorder in instances wherein only asubset of the cells within a tissue or a population of cells is thoughtto be relevant to the disorder.

Third, the sequences of the identified genes can be used, utilizingstandard techniques, to place the genes onto genetic maps, e.g., mouse(Copeland, N. G. and Jenkins, N. A., 1991, Trends in Genetics 7:113-118)and human genetic maps (Cohen, D., et al., 1993, Nature 366:698-701).Such mapping information can yield information regarding the genes'importance to human disease by, for example, identifying genes which mapwithin genetic regions to which known genetic TH cellsubpopulation-related disorders map. Such regions include, for example,the mouse Scl-1 locus, which is suspected to be involved inLeishmaniasis, or the human 5q31.1 chromosomal region which contains oneor more loci thought to regulate IgE production in a nonantigen-specificfashion, and can, therefore, be involved in allergy, a TH2-like-relateddisorder (Marsh, D. et al., 1994, Science 264:1152-1156).

Fourth, the biological function of the identified genes can be moredirectly assessed by utilizing relevant in vivo and in vitro systems. Invivo systems can include, but are not limited to, animal systems whichnaturally exhibit the symptoms of immune disorders, or ones which havebeen engineered to exhibit such symptoms. Further, such systems caninclude systems for the further characterization of the cell typedifferentiation and effector function, and can include, but are notlimited to transgenic animal systems such as those described, above, inSection 5.1.1.1, and Section 5.7.1, below. In vitro systems can include,but are not limited to, cell-based systems comprising, for example, TH1or TH2 cell types. The TH subpopulation cells can be wild type cells, orcan be non-wild type cells containing modifications known or suspectedof contributing to the TH cell subpopulation-related disorder ofinterest. Such systems are discussed in detail, below, in Section 5.7.2.

In further characterizing the biological function of the identifiedgenes, the expression of these genes can be modulated within the in vivoand/or in vitro systems, i.e., either overexpressed or underexpressedin, for example, transgenic animals and/or cell lines, and itssubsequent effect on the system can then be assayed. Alternatively, theactivity of the product of the identified gene can be modulated byeither increasing or decreasing the level of activity in the in vivoand/or in vitro system of interest, and its subsequent effect thenassayed.

The information obtained through such characterizations can suggestrelevant methods for the treatment or control of immune disorders, suchas TH cell subpopulation-related disorders, involving the gene ofinterest. For example, relevant treatment can include not only amodulation of gene expression and/or gene product activity, but can alsoinclude a selective depletion or stimulation of the TH cellsubpopulation of interest. Characterization procedures such as thosedescribed herein can indicate where such modulation should be positiveor negative. As used herein, "positive modulation" refers to an increasein gene expression or activity of the gene or gene product of interest,or to a stimulation of a TH cell subpopulation, relative to thatobserved in the absence of the modulatory treatment. "Negativemodulation", as used herein, refers to a decrease in gene expression oractivity, or a depletion of a TH cell subpopulation, relative to thatobserved in the absence of the modulatory treatment. "Stimulation" and"depletion" are as defined, above, in Section 3. Methods of treatmentare discussed, below, in Section 5.9.

5.4 DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

Differentially expressed genes such as those identified in Section5.1.1, above, and pathway genes, such as those identified in Section5.2, above, are described herein.

The differentially expressed and pathway genes of the invention arelisted below, in Table 1. Differentially expressed gene sequences areshown in FIGS. 2, 4A, 9 and 12-15, 17 and 22. The nucleotide sequencesidentified via differential display analysis are referred to herein asband 10, 54, 57, 102, 103, 105, 106, 161 and 200. The genescorresponding to these sequences are referred to herein as the 10, 54,57, 102, 103, 106, 161 and 200 genes, respectively. Table 1 listsdifferentially expressed genes identified through, for example, theparadigms discussed, above, in Section 5.1.1.1, and below, in theExamples presented in Sections 6-8.

Table 1 summarizes information regarding the further characterization ofsuch genes. Table 2 lists E. coli clones, deposited with theAgricultural Research Service Culture Collection (NRRL), which containsequences found within the genes of Table 1.

In Table 1, the column headed "Diff. Exp." details the differentialexpression characteristic by which the sequence has been identified.Under this column, "TH Inducible", refers to those cases wheredifferential expression arises upon exposure of the cell type ofinterest to an agent capable of bringing about TH cell stimulation oractivation. These sequences, therefore, are differentially expressed inundifferentiated, partially or fully differentiated TH cells, and thegenes corresponding to these sequences are expressed in both TH1 and TH2cell subpopulations.

"TH1", under this column, refers to a sequence corresponding to a geneexpressed preferentially in mature, fully differentiated TH1 cellsrelative to TH2 cells. "TH2", under this column, refers to a sequencecorresponding to a gene preferentially expressed in mature, fullydifferentiated TH2 cell subpopulations relative to TH1 cellsubpopulations. Preferential expression can be qualitative orquantitative, as described, above, in Section 5.1.

Tissue expression patterns are also summarized in Table 1. The columnheaded "Tissue/Cell Dist." lists tissues and/or cell types in whichexpression of the gene has been tested and whether expression of thegene within a given tissue or cell type has been observed. Specifically,"+" indicates detectable mRNA from the gene of interest, while "-"refers to no detectable mRNA from the gene of interest. Unless otherwisenoted, "+" and "-" refer to all samples of a given tissue or cell typetested. "Detectable", as used herein, is as described, above, in Section5.1.

Additionally, the physical locus to which the gene maps on the humanand/or mouse chromosome map is indicated in the column headed "Locus".Further, in instances wherein the genes correspond to genes known to befound in nucleic acid databases, references (i.e., citations and/or genenames) to such known genes are listed in the column headed "Ref.".

The genes listed in Table 1 can be obtained using cloning methods wellknown to those of skill in the art, and include but are not limited tothe use of appropriate probes to detect the genes within an appropriatecDNA or gDNA (genomic DNA) library. (See, for example, Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, which is incorporated herein by reference in itsentirety.) Probes for the sequences reported herein can be obtaineddirectly from the isolated clones deposited with the NRRL, as indicatedin Table 2, below. Alternatively, oligonucleotide probes for the genescan be synthesized based on the DNA sequences disclosed herein in FIGS.2, 4A, 9, 12-15, 17 and 22. With respect to the previously reportedgenes, synthetic oligonucleotides can be synthesized or produced basedon the sequences provided for the previously known genes described inthe following references: granzyme A, Hanukah factor: Masson, D. et al.,1986, FEBS Lett. 208:84-88; Masson, D. et al., 1986, EMBO J.5:1595-1600; Gershenfeld, H. K. and Weissman, I. L., 1986, Science232:854-858; ST-2, T1, Fit-1: Klemenz, R. et al., 1989, Proc. Natl.Acad. Sci. USA 86:5708-5712; Tominaga, S., 1989, FEBS Lett. 258:301-301;Werenskiold, A. K. et al., 1989, Mol. Cell. Biol. 9:5207-5214; Tominaga,S. et al., 1992, Biochem. Biophys. Acta. 1171:215-218; Werenskiold, A.K., 1992, Eur. J. Biochem. 204:1041-1047; Yanagisawa, K. et al., 1993,FEBS Lett. 318:83-87; and Bergers, G. et al., 1994, EMBO J.13:1176-1188.

The probes can be used to screen cDNA libraries prepared from anappropriate cell or cell line in which the gene is transcribed.Appropriate cell lines can include, for example, Dorris, AE7, D10.G4,DAX, D1.1 and CDC25 cell lines. In addition, purified primary naive Tcells derived from either transgenic or non-transgenic strains can beused. Alternatively, the genes described herein can be cloned from acDNA library constructed from, for example, NIH 3T3 cell lines stablytransfected with the Ha-ras(EJ) gene, 5C10 cells, and peripheral bloodlymphocytes.

                  TABLE 1                                                         ______________________________________                                        DIFFERENTIALLY EXPRESSED AND PATHWAY GENES                                      Gene      Diff. Exp.                                                                              Tissue/Cell Dist.                                                                        Locus Ref                                    ______________________________________                                        102     TH2       TH2 Specific       ref 1                                      103           TH2               (+)                                                                              ref 2                                                                      TH2                                                                           (-)                                                                           Lymph Node;                                                                   Spleen;                                                                       Thymus;                                                                       Brain;                                                                        Lung;                                                                         Bone Marrow;                                                                  Heart;                                                                        Spleen                                         10            TH                (+)                       See                              Inducible         Spleen;                      FIG. 11                                             TH1;                                                                       TH2.                                                                          (-)                                                                           Liver;                                                                        Brain;                                                                        Thymus;                                                                       Bone Marrow;                                                                  Heart;                                                                        Lymph Node.                                    57            TH                (+)                                                        Inducible         TH1;                                                                          TH2;                                                                          Spleen                                        105           TH1               (+)                                                                           TH1;                                                                          Spleen                                        106           TH1               (+)                                                                           TH1;                                                                          Thymus;                                                                       Spleen                                        161           Subset            (+)                                                         Specific.sup.3     Spleen                                                                       (-)                                                                           Thymus                                        200           TH1               (+)                                                                           TH1                                            54            TH1               (+) TH1;                                                                     spleen;                                                                       testis; uterus                                                                (-)brain;                                                                     heart; kidney;                                                                liver; muscle                               ______________________________________                                         .sup.1 Masson, D. et al., 1986, FEBS Lett. 208:84-88; Masson, D. et al.,      1986, EMBO J. 5:1595-1600; Gershenfeld, H. K. and Weissman, I. L., 1986,      Science 232:854-858.                                                          .sup.2 Klemenz, R. et al., 1989, Proc. Natl. Acad. Sci. USA 86:5708-5712;     Tominaga, S., 1989, FEBS Lett. 258:301-301; Werenskiold, A. K. et al.,        1989, Mol. Cell. Biol. 9:5207-5214; Tominaga, S. et al., 1992, Biochem.       Biophys. Acta. 1171:215-218; Werenskiold, A. K., 1992, Eur. J. Biochem.       204:1041-1047; Yanagisawa, K. et al., 1993, FEBS Lett. 318:83-87; Bergers     G. et al., 1994, EMBO J. 13:1176-1188.                                        .sup.3 Band 161 expression has been observed in either TH1 or TH2 cell        subpopulations, but has not been found, simultaneously, in both TH1 and       TH2 cell subpopulations.                                                 

Table 2, below, lists isolated E. coli clones which contain sequenceswithin the novel genes listed in Table 1.

                  TABLE 2                                                         ______________________________________                                        GENE          CLONE                                                           ______________________________________                                         10           10-C                                                               10                        10-X                                                57                        57-E                                               105                       105-A                                               106                       106-H                                               161                       161-G                                               200                       200-O                                               200                       DH10B(Zip) ™ containing 200-P                    200                       200-AF                                               54                        54-C                                             ______________________________________                                    

As used herein, "differentially expressed gene" (i.e. target andfingerprint gene) or "pathway gene" refers to (a) a gene containing: atleast one of the DNA sequences disclosed herein (as shown in FIGS. 2,4A, 9, 12-15, 17 and 22), or contained in the clones listed in Table 2,as deposited with the NRRL; (b) any DNA sequence that encodes the aminoacid sequence encoded by: the DNA sequences disclosed herein (as shownin FIGS. 2, 4A, 9, 12-15, 17 and 22), contained in the clones, listed inTable 2, as deposited with the NRRL or contained within the codingregion of the gene to which the DNA sequences disclosed herein (as shownin FIGS. 2, 4A, 9, 12-15, 17 and 22) or contained in the clones listedin Table 2, as deposited with the NRRL, belong; (c) any DNA sequencethat hybridizes to the complement of: the coding sequences disclosedherein (as shown in FIGS. 2, 4A, 9, 12-15, 17 and 22), contained inclones listed in Table 2, as deposited with the NRRL, or containedwithin the coding region of the gene to which the DNA sequencesdisclosed herein (as shown in FIGS. 2, 4A, 9, 12-15, 17 and 22) orcontained in the clones listed in Table 2, as deposited with the NRRL,belong, under highly stringent conditions, e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 2.10.3), and encodes a gene product functionally equivalentto a gene product encoded by a gene of (a), above; and/or (d) any DNAsequence that hybridizes to the complement of: the coding sequencesdisclosed herein, (as shown in FIGS. 2, 4A, 9, 12-15, 17 and 22)contained in the clones listed in Table 2, as deposited with the NRRL orcontained within the coding region of the gene to which DNA sequencesdisclosed herein (as shown in FIGS. 2, 4A, 9, 12-15, 17 and 22) orcontained in the clones, listed in Table 2, as deposited with the NRRL,belong, under less stringent conditions, such as moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet which still encodes a gene product functionallyequivalent to a gene product encoded by a gene of (a), above.

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, theDNA sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions can be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides ("oligos"), highly stringentconditions can refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules can act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target, fingerprint, and/orpathway gene nucleic acid sequences. Further, such sequences can be usedas part of ribozyme and/or triple helix sequences, also useful fortarget gene regulation. Still further, such molecules can be used ascomponents of diagnostic methods whereby the presence of, orpredisposition to, an immune disorder, e.g., TH cellsubpopulation-related disorder, can be detected.

The invention also encompasses (a) DNA vectors that contain any of theforegoing coding sequences and/or their complements (i.e., antisense);(b) DNA expression vectors that contain any of the foregoing codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences; and (c) genetically engineeredhost cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Theinvention includes fragments of any of the DNA sequences disclosedherein.

In addition to the gene sequences described above, homologs of thesegene sequences as can, for example, be present in other species,preferably human in instances wherein the above-described gene sequencesare not human gene sequences, can be identified and isolated, withoutundue experimentation, by molecular biological techniques well known inthe art. Further, there can exist genes at other genetic loci within thegenome of the same species that encode proteins which have extensivehomology to one or more domains of such gene products. These genes canalso be identified via similar techniques.

For example, the isolated differentially expressed gene sequence can belabeled and used to screen a cDNA library constructed from mRNA obtainedfrom the organism of interest. Hybridization conditions will be of alower stringency when the cDNA library was derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. Alternatively, the labeled fragment can be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. Such low stringency conditions willbe well known to those of skill in the art, and will vary predictablydepending on the specific organisms from which the library and thelabeled sequences are derived. For guidance regarding such conditionssee, for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, (Green Publishing Associates andWiley Interscience, N.Y.).

Further, a previously unknown differentially expressed or pathwaygene-type sequence can be isolated by performing PCR using twodegenerate oligonucleotide primer pools designed on the basis of aminoacid sequences within the gene of interest. The template for thereaction can be cDNA obtained by reverse transcription of mRNA preparedfrom human or non-human cell lines or tissue known or suspected toexpress a differentially expressed or pathway gene allele. The PCRproduct can be subcloned and sequenced to insure that the amplifiedsequences represent the sequences of a differentially expressed orpathway gene-like nucleic acid sequence.

The PCR fragment can then be used to isolate a full length cDNA clone bya variety of methods. For example, the amplified fragment can be used toscreen a bacteriophage cDNA library. Alternatively, the labeled fragmentcan be used to screen a genomic library.

PCR technology can also be utilized to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5' end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid can then be "tailed" with guanines using a standardterminal transferase reaction, the hybrid can be digested with RNAase H,and second strand synthesis can then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment can easily beisolated. For a review of cloning strategies which can be used, seee.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, (Green Publishing Associates and WileyInterscience, N.Y.).

In cases where the differentially expressed or pathway gene identifiedis the normal, or wild type, gene, this gene can be used to isolatemutant alleles of the gene. Such an isolation is preferable in processesand disorders which are known or suspected to have a genetic basis.Mutant alleles can be isolated from individuals either known orsuspected to have a genotype which contributes to TH cellsubpopulation-disorder related symptoms. Mutant alleles and mutantallele products can then be utilized in the therapeutic and diagnosticassay systems described below.

A cDNA of a mutant gene can be isolated, for example, by using PCR, atechnique which is well known to those of skill in the art. In thiscase, the first cDNA strand can be synthesized by hybridizing a oligo-dToligonucleotide to mRNA isolated from tissue known to, or suspected of,being expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5' end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. The normal gene or any suitable fragmentthereof can then be labeled and used as a probed to identify thecorresponding mutant allele in the library. The clone containing thisgene can then be purified through methods routinely practiced in theart, and subjected to sequence analysis as described, above, in thisSection.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. In this manner, gene products made by theputatively mutant tissue can be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal gene product, as described, below, in Section 5.6.(For screening techniques, see, for example, Harlow, E. and Lane, eds.,1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, ColdSpring Harbor.) In cases where the mutation results in an expressed geneproduct with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described in this Section, above.

5.5 DIFFERENTIALLY EXPRESSED AND PATHWAY GENE PRODUCTS

Differentially expressed and pathway gene products include thoseproteins encoded by the differentially expressed and pathway genescorresponding to the gene sequences described in Section 5.4, above, as,for example, the peptides listed in FIGS. 9, 17 and 22.

In addition, differentially expressed and pathway gene products caninclude proteins that represent functionally equivalent gene products.Such an equivalent differentially expressed or pathway gene product cancontain deletions, additions or substitutions of amino acid residueswithin the amino acid sequence encoded by the differentially expressedor pathway gene sequences described, above, in Section 5.4, but whichresult in a silent change, thus producing a functionally equivalentdifferentially expressed or pathway gene product. Amino acidsubstitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. "Functionallyequivalent", as utilized herein, refers to a protein capable ofexhibiting a substantially similar in vivo activity as the endogenousdifferentially expressed or pathway gene products encoded by thedifferentially expressed or pathway gene sequences described in Section5.4, above. Alternatively, when utilized as part of assays such as thosedescribed, below, in Section 5.3, "functionally equivalent" can refer topeptides capable of interacting with other cellular or extracellularmolecules in a manner substantially similar to the way in which thecorresponding portion of the endogenous differentially expressed orpathway gene product would.

The differentially expressed or pathway gene products can be produced bysynthetic techniques or via recombinant DNA technology using techniqueswell known in the art. Thus, methods for preparing the differentiallyexpressed or pathway gene polypeptides and peptides of the invention aredescribed herein. First, the polypeptides and peptides of the inventioncan be synthesized or prepared by techniques well known in the art. See,for example, Creighton, 1983, "Proteins: Structures and MolecularPrinciples", W.H. Freeman and Co., N.Y., which is incorporated herein byreference in its entirety. Peptides can, for example, be synthesized ona solid support or in solution.

Alternatively, recombinant DNA methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining differentially expressed or pathway gene protein codingsequences and appropriate transcriptional/translational control signals.These methods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. which is incorporated by reference herein in theirentirety, and Ausubel, 1989, supra. Alternatively, RNA capable ofencoding differentially expressed or pathway gene protein sequences canbe chemically synthesized using, for example, synthesizers. See, forexample, the techniques described in "Oligonucleotide Synthesis", 1984,Gait, M. J. ed., IRL Press, Oxford, which is incorporated by referenceherein in its entirety.

A variety of host-expression vector systems can be utilized to expressthe differentially expressed or pathway gene coding sequences of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest can be produced and subsequently purified,but also represent cells which can, when transformed or transfected withthe appropriate nucleotide coding sequences, exhibit the differentiallyexpressed or pathway gene protein of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing differentiallyexpressed or pathway gene protein coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the differentially expressed or pathway gene proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (eq., baculovirus) containing the differentiallyexpressed or pathway gene protein coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingdifferentially expressed or pathway gene protein coding sequences; ormammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for thedifferentially expressed or pathway gene protein being expressed. Forexample, when a large quantity of such a protein is to be produced, forthe generation of antibodies or to screen peptide libraries, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified can be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which thedifferentially expressed or pathway gene protein coding sequence can beligated individually into the vector in frame with the lacZ codingregion so that a fusion protein is produced; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors can alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene protein can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The differentially expressed or pathwaygene coding sequence can be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of differentially expressed or pathway gene codingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed, (e.g., see Smith et al., 1983, J. Viol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the differentially expressed or pathway gene coding sequence ofinterest can be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene can then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing differentially expressedor pathway gene protein in infected hosts, (e.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals can also be required for efficient translation of inserteddifferentially expressed or pathway gene coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherean entire differentially expressed or pathway gene, including its owninitiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals can be needed. However, in cases where only a portion of thedifferentially expressed or pathway gene coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe differentially expressed or pathway gene protein can be engineered.Rather than using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells can be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method can advantageously be used to engineer cell lineswhich express the differentially expressed or pathway gene protein. Suchengineered cell lines can be particularly useful in screening andevaluation of compounds that affect the endogenous activity of thedifferentially expressed or pathway gene protein.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

When used as a component in assay systems such as those describedherein, the differentially expressed or pathway gene protein can belabeled, either directly or indirectly, to facilitate detection of acomplex formed between the differentially expressed or pathway geneprotein and a test substance. Any of a variety of suitable labelingsystems can be used including but not limited to radioisotopes such as¹²⁵ I; enzyme labelling systems that generate a detectable calorimetricsignal or light when exposed to substrate; and fluorescent labels.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to either a differentially expressedor pathway gene product. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by an Fab expression library.

Where recombinant DNA technology is used to produce the differentiallyexpressed or pathway gene protein for such assay systems, it can beadvantageous to engineer fusion proteins that can facilitate labeling(either direct or indirect), immobilization, solubility and/ordetection.

Fusion proteins, which can facilitate solubility and/or expression, andcan increase the blood half-life of the protein, can include, but arenot limited to soluble Ig-tailed fusion proteins. Methods forengineering such soluble Ig-tailed fusion proteins are well known tothose of skill in the art. See, for example U.S. Pat. No. 5,116,964,which is incorporated herein by reference in its entirety.

Among the soluble Ig-tailed fusion proteins which can be produced aresoluble Ig-tailed fusion proteins containing 103 gene products, 200 geneproducts or 10 gene products. The 103 gene product or 200 gene containedwithin such fusion proteins can comprise, respectively, for example, the103 gene extracellular domain or portions, preferably ligand-bindingportions, thereof, or the 200 gene extracellular domain or portions,preferably ligand-binding portions, thereof. The 10 gene productcontained within such fusion proteins can comprise, for example, one ormore of the extracellular domains or portions, preferably ligand-bindingportions, of the seven transmembrane domain sequence motif.

The amino acid sequences of the 103 gene products are known. (See, forexample, Klemenz, R. et al., 1989, Proc. Natl. Acad. Sci. USA86:5708-5712; Tominaga, S., 1989, FEBS Lett. 258:301--301; Werenskiold,A. K. et al., 1989, Mol. Cell. Biol.9:5207-5214; Tominaga, S. et al.,1992, Biochem. Biophys. Acta. 1171:215-218; Werenskiold, A. K., 1992,Eur. J. Biochem. 204:1041-1047; Yanagisawa, K. et al., 1993, FEBS Lett.318:83-87; Bergers, G. et al., 1994, EMBO J. 13:1176-1188.) Further, asindicated in FIG. 4B, the amino acid residues which delineate theextracellular, transmembrane and cytoplasmic domains of the 103 geneproducts are also known. Therefore, by utilizing well known techniques,one of skill in the art would readily be capable of producing suchsoluble Ig-tailed 103 gene product fusion proteins.

The 280 amino acid 200 gene product (FIG. 17, SEQ ID NO:10)extracellular, transmembrane and cytoplasmic domains have beenelucidated and can be utilized in, for example, the construction of 200gene product-Ig fusion proteins. Specifically, the 200 geneextracellular domain contains amino acid residue 1 to approximatelyamino acid 191, its transmembrane domain contains from approximatelyamino acid residue 192 to amino acid residue 214, and its cytoplasmicdomain contains from approximately amino acid residue 215 to amino acidresidue 280. Given the elucidation of these domains, one of skill in theart would readily be capable of producing soluble Ig-tailed 200 geneproduct fusion proteins. The Example presented, below, in Section 9describes the construction of a 200 gene product-Ig fusion protein.

The 338 amino acid residue 10 gene product (FIG. 9, SEQ ID NO:9)extracellular domains include 10 gene product amino acid residues fromapproximately amino acid residue 1 to 19, amino acid residue 74 to 87,amino acid residue 153 to 187 and amino acid residue 254 to 272. Thus,such 10 gene product domain information can be used, in conjunction withwell-known techniques, such that one of skill in the art can readily becapable of producing soluble Ig-tailed 10 gene fusion proteinscomprising one or more 10 gene product extra-cellular domain regions andan Ig tail.

5.6. ANTIBODIES SPECIFIC FOR DIFFERENTIALLY EXPRESSED OR PATHWAY GENEPRODUCTS

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed or pathwaygene product epitopes. Such antibodies can include, but are not limitedto, polyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. The Ig tails of such antibodies can be modified toreduce complement activation and Fc binding. (See, for example, EuropeanPatent No. 239400 B1, Aug. 3, 1994).

Such antibodies can be used, for example, in the detection of afingerprint, target, or pathway gene product in a biological sample, andcan be used as part of diagnostic techniques. Alternatively, suchantibodies can be utilized as part of an immune disorder treatmentmethod, as described, below, in Section 5.9. For example, the antibodiescan be used to modulate target gene activity, can be used to modulate THcell subpopulation differentiation, maintenance and/or effectorfunction, or, in the case of antibodies directed to cell surfaceepitopes, can be used to isolate a TH cell subpopulation of interest,for either depletion or augmentation purposes.

For the production of antibodies to a differentially expressed orpathway gene, various host animals can be immunized by injection with adifferentially expressed or pathway gene protein, or a portion thereof.Such host animals can include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants can be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, can be immunized by injection withdifferentially expressed or pathway gene product supplemented withadjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies can be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention can be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of "chimericantibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454; U.S. Pat. No. 4,816,567) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) and for making humanized monoclonalantibodies (U.S. Pat. No. 5,225,539, which is incorporated herein byreference in its entirety) can be utilized to produceanti-differentially expressed or anti-pathway gene product antibodies.

Antibody fragments which recognize specific epitopes can be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab')₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

5.7. CELL-AND ANIMAL-BASED MODEL SYSTEMS

Described herein are cell- and animal-based systems which act as modelsfor immune disorders and for models of TH cell subpopulationdifferentiation, maintenance, and/or effector function. These systemscan be used in a variety of applications. For example, the animal-basedmodel systems can be utilized to identify differentially expressed genesvia the in vivo paradigm described, above, in Section 5.1.1.1. Cell- andanimal-based model systems can also be used to further characterizedifferentially expressed and pathway genes, as described, above, inSection 5.3. Such further characterization can, for example, indicatethat a differentially expressed gene is a target gene. Second, suchassays can be utilized as part of screening strategies designed toidentify compounds which are capable of ameliorating TH cellsubpopulation-related disorder symptoms, as described, below. Thus, theanimal- and cell-based models can be used to identify drugs,pharmaceuticals, therapies and interventions which can be effective intreating immune disorders such as TH cell subpopulation-relateddisorders. In addition, as described in detail, below, in Section5.10.1, such animal models can be used to determine the LD₅₀ and theED₅₀ in animal subjects, and such data can be used to determine the invivo efficacy of potential immune disorder treatments.

5.7.1 ANIMAL-BASED SYSTEMS

Animal-based model systems of TH cell subpopulation-related disorderscan include both non-recombinant animals as well as recombinantlyengineered transgenic animals.

Animal models for TH cell subpopulation-related disorders can include,for example, genetic models. For example, such animal models can includeLeishmania resistance models, experimental allergic encephalomyelitismodels and (BALB/c Cr×DBA/2Cr) F1 mice. These latter mice develop afatal disseminated disease by systemic infection with virulent Candidaalbicans associated with strong TH2-like responses. Additionally, wellknown mouse models for asthma can be utilized to study the ameliorationof symptoms caused by a TH2-like response. (See, for example, Lukacs, N.W. et al., 1994, Am. J. Resp. Cell Mol. Biol. 10:526-532; Gavett, S. H.et al., 1994, Am. J. Resp. Cell Mol. Biol. 10:587-593.) Further, theanimal model, murine acquired immunodeficiency syndrome (MAIDS;Kanagawa, B. et al., 1993, Science 262:240; Makino, M. et al., 1990, J.Imm. 144:4347) can be used for such studies.

Alternatively, such well known animal models as SCIDhu mice (see forexample, Keneshima, H. et al., 1994, Curr. Opin. Imm. 6:327-333) whichrepresents an in vivo model of the human hematolymphoid system, can beutilized. Further, the RAG-2-deficient blastocyst complementationtechnique (Chen, J. et al., 1993, Proc. Natl. Acad. Sci. USA90:4528-4532; Shinkai, Y. et al., 1992, Cell 68:855-867) can be utilizedto produce mice containing, for example, humanized lymphocytes and/orwhich express target gene sequences. Still further, targeting techniquesdirected specifically to T cells, for example, the technique of Gu etal. (Gu, H. et al., 1994, Science 265:103-106) can be utilized toproduce animals containing transgenes in only T cell populations.

Animal models exhibiting TH cell subpopulation-related disorder-likesymptoms can be engineered by utilizing, for example, target genesequences such as those described, above, in Section 5.4, in conjunctionwith techniques for producing transgenic animals that are well known tothose of skill in the art. For example, target gene sequences can beintroduced into, and overexpressed in, the genome of the animal ofinterest, or, if endogenous target gene sequences are present, they caneither be overexpressed or, alternatively, can be disrupted in order tounderexpress or inactivate target gene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence can be ligated to a regulatory sequence whichis capable of driving gene expression in the animal and cell type ofinterest. Such regulatory regions will be well known to those of skillin the art, and can be utilized in the absence of undue experimentation.

For underexpression of an endogenous target gene sequence, such asequence can be isolated and engineered such that when reintroduced intothe genome of the animal of interest, the endogenous target gene alleleswill be inactivated. Preferably, the engineered target gene sequence isintroduced via gene targeting such that the endogenous target sequenceis disrupted upon integration of the engineered target gene sequenceinto the animal's genome. Gene targeting is discussed, below, in thisSection.

Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, squirrels, monkeys, and chimpanzees can be used togenerate animal models of TH cell subpopulation-related disorders.

Any technique known in the art can be used to introduce a target genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. (See,for example, techniques described by Jakobovits, 1994, Curr. Biol.4:761-763.) The transgene can be integrated as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene can also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art.

When it is desired that the target gene transgene be integrated into thechromosomal site of the endogenous target gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous targetgene of interest are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of, the nucleotide sequence of the endogenous target gene.The transgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous gene of interest in only thatcell type, by following, for example, the teaching of Gu et al. (Gu, H.et al., 1994, Science 265:103-106). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art.

Once transgenic animals have been generated, the expression of therecombinant target gene and protein can be assayed utilizing standardtechniques. Initial screening can be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals canalso be assessed using techniques which include but are not limited toNorthern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of targetgene-expressing tissue, can also be evaluated immunocytochemically usingantibodies specific for the target gene transgene gene product ofinterest.

The target gene transgenic animals that express target gene mRNA ortarget gene transgene peptide (detected immunocytochemically, usingantibodies directed against target gene product epitopes) at easilydetectable levels can then be further evaluated to identify thoseanimals which display characteristic TH cell subpopulation-relateddisorder-like symptoms, or exhibit characteristic TH cell subpopulationdifferentiation phenotypes. TH1-like-related disorder symptoms caninclude, for example, those associated with chronic inflammatorydiseases and disorders, such as Crohn's disease, reactive arthritis,including Lyme disease, insulin-dependent diabetes, organ-specificautoimmunity, including multiple sclerosis, Hashimoto's thyroiditis andGrave's disease, contact dermatitis, psoriasis, graft rejection, graftversus host disease and sarcoidosis. TH2-like-related disorder symptomscan include, those associated with atopic conditions, such as asthma andallergy, including allergic rhinitis, gastrointestinal allergies,including food allergies, eosinophilia, conjunctivitis, glomerularnephritis, certain pathogen susceptibilities such as helminthic (e.g.,leishmaniasis) and certain viral infections, including HIV, andbacterial infections, including tuberculosis and lepromatous leprosy.

Additionally, specific cell types within the transgenic animals can beanalyzed and assayed for cellular phenotypes characteristic of TH cellsubpopulation-related disorders. Such cellular phenotypes can include,for example, differential cytokine expression characteristic of the THcell subpopulation of interest. Further, such cellular phenotypes caninclude an assessment of a particular cell type's fingerprint pattern ofexpression and its comparison to known fingerprint expression profilesof the particular cell type in animals exhibiting specific TH cellsubpopulation-related disorders. Such transgenic animals serve assuitable model systems for TH cell-related disorders.

Once target gene transgenic founder animals are produced (i.e., thoseanimals which express target gene proteins in cells or tissues ofinterest, and which, preferably, exhibit symptoms of TH cellsubpopulation-related disorders), they can be bred, inbred, outbred, orcrossbred to produce colonies of the particular animal. Examples of suchbreeding strategies include but are not limited to: outbreeding offounder animals with more than one integration site in order toestablish separate lines; inbreeding of separate lines in order toproduce compound target gene transgenics that express the target genetransgene of interest at higher levels because of the effects ofadditive expression of each target gene transgene; crossing ofheterozygous transgenic animals to produce animals homozygous for agiven integration site in order to both augment expression and eliminatethe possible need for screening of animals by DNA analysis; crossing ofseparate homozygous lines to produce compound heterozygous or homozygouslines; breeding animals to different inbred genetic backgrounds so as toexamine effects of modifying alleles on expression of the target genetransgene and the development of TH cell subpopulation-relateddisorder-like symptoms. One such approach is to cross the target genetransgenic founder animals with a wild type strain to produce an F1generation that exhibits TH cell subpopulation-related disorder-likesymptoms, such as those described above. The F1 generation can then beinbred in order to develop a homozygous line, if it is found thathomozygous target gene transgenic animals are viable.

5.7.2. CELL-BASED ASSAYS

Cells that contain and express target gene sequences which encode targetgene protein, and, further, exhibit cellular phenotypes associated witha TH cell subpopulation-related disorder of interest, can be utilized toidentify compounds that exhibit an ability to ameliorate TH cellsubpopulation-related disorder symptoms. Cellular phenotypes which canindicate an ability to ameliorate TH cell subpopulation-related disordersymptoms can include, for example, an inhibition or potentiation ofcytokine or cell surface marker expression associated with the TH cellsubpopulation of interest, or, alternatively, an inhibition orpotentiation of specific TH cell subpopulations.

Further, the fingerprint pattern of gene expression of cells of interestcan be analyzed and compared to the normal, non-TH cellsubpopulation-related disorder fingerprint pattern. Those compoundswhich cause cells exhibiting TH cell subpopulation-related disorder-likecellular phenotypes to produce a fingerprint pattern more closelyresembling a normal fingerprint pattern for the cell of interest can beconsidered candidates for further testing regarding an ability toameliorate TH cell subpopulation-related disorder symptoms.

Cells which can be utilized for such assays can, for example, includenon-recombinant cell lines, such as Dorris, AE7, D10.G4, DAX, D1.1 andCDC25 cell lines. In addition, purified primary naive T cells derivedfrom either transgenic or non-transgenic strains can also be used.

Further, cells which can be used for such assays can also includerecombinant, transgenic cell lines. For example, the TH cellsubpopulation-related disorder animal models of the invention,discussed, above, in Section 5.7.1, can be used to generate, forexample, TH1-like and/or TH2-like cell lines that can be used as cellculture models for the disorder of interest. While primary culturesderived from TH cell subpopulation-related disorder transgenic animalscan be utilized, the generation of continuous cell lines is preferred.For examples of techniques which can be used to derive a continuous cellline from the transgenic animals, see Small et al., 1985, Mol. CellBiol. 5:642-648.

Alternatively, cells of a cell type known to be involved in TH cellsubpopulation-related disorders can be transfected with sequencescapable of increasing or decreasing the amount of target gene expressionwithin the cell. For example, target gene sequences can be introducedinto, and overexpressed in, the genome of the cell of interest, or, ifendogenous target gene sequences are present, they can either beoverexpressed or, alternatively, can be disrupted in order tounderexpress or inactivate target gene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence can be ligated to a regulatory sequence whichis capable of driving gene expression in the cell type of interest. Suchregulatory regions will be well known to those of skill in the art, andcan be utilized in the absence of undue experimentation.

For underexpression of an endogenous target gene sequence, such asequence can be isolated and engineered such that when reintroduced intothe genome of the cell type of interest, the endogenous target genealleles will be inactivated. Preferably, the engineered target genesequence is introduced via gene targeting such that the endogenoustarget sequence is disrupted upon integration of the engineered targetgene sequence into the cell's genome. Gene targeting is discussed,above, in Section 5.7.1.

Transfection of target gene sequence nucleic acid can be accomplished byutilizing standard techniques. See, for example, Ausubel, 1989, supra.Transfected cells should be evaluated for the presence of therecombinant target gene sequences, for expression and accumulation oftarget gene mRNA, and for the presence of recombinant target geneprotein production. In instances wherein a decrease in target geneexpression is desired, standard techniques can be used to demonstratewhether a decrease in endogenous target gene expression and/or in targetgene product production is achieved.

5.8. SCREENING ASSAYS FOR COMPOUNDS THAT INTERACT WITH THE TARGET GENEPRODUCT

The following assays are designed to identify compounds that bind totarget gene products, bind to other cellular proteins that interact witha target gene product, and to compounds that interfere with theinteraction of the target gene product with other cellular proteins. Forexample, in the cases of 10, 103 and 200 gene products, which are or arepredicted to be transmembrane receptor-type proteins, such techniquescan identify ligands for such receptors. A 103 gene product ligand can,for example, act as the basis for amelioration of such TH2-like-specificdisorders as asthma or allergy, given that gene 103 expression isTH2-specific. A 200 gene product ligand can, for example, act as thebasis for amelioration of TH1-like-specific disorders. A 10 gene productligand can, for example, act as the basis for amelioratoin of a widerange of T cell disorders, given the TH inducible nature of it geneexpression pattern.

Compounds can include, but are not limited to, other cellular proteins.Further, such compounds can include, but are not limited to, peptidessuch as, for example, soluble peptides, including, but not limited to,Ig-tailed fusion peptides, comprising extracellular portions of targetgene product transmembrane receptors, and members of random peptidelibraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghten, R. et al., 1991, Nature 354:84-86) made of D-and/orL-configuration amino acids, phosphopeptides (including but not limitedto members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab')₂ and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can beuseful, for example, in elaborating the biological function of thetarget gene product, and for ameliorating the symptoms of immunedisorders. In instances, for example, in which a TH cellsubpopulation-related disorder situation results from a lower overalllevel of target gene expression, target gene product, and/or target geneproduct activity in a cell or tissue involved in such a disorder,compounds that interact with the target gene product can include oneswhich accentuate or amplify the activity of the bound target geneprotein. Such compounds would bring about an effective increase in thelevel of target gene activity, thus ameliorating symptoms. In instanceswhereby mutations within the target gene cause aberrant target geneproteins to be made which have a deleterious effect that leads to a THcell subpopulation-related disorder, or, alternatively, in instanceswhereby normal target gene activity is necessary for a TH cellsubpopulation-related disorder to occur, compounds that bind target geneprotein can be identified that inhibit the activity of the bound targetgene protein. Assays for identifying additional compounds as well as fortesting the effectiveness of compounds, identified by, for example,techniques, such as those described in Section 5.8.1-5.8.3, arediscussed, below, in Section 5.8.4.

5.8.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO A TARGETGENE PRODUCT

In vitro systems can be designed to identify compounds capable ofbinding the target gene products of the invention. Compounds identifiedcan be useful, for example, in modulating the activity of wild typeand/or mutant target gene products, can be useful in elaborating thebiological function of target gene products, can be utilized in screensfor identifying compounds that disrupt normal target gene productinteractions, or can in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to thetarget gene product involves preparing a reaction mixture of the targetgene product and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringtarget gene product or the test substance onto a solid phase anddetecting target gene product/test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the target gene product can be anchored onto a solid surface,and the test compound, which is not anchored, can be labeled, eitherdirectly or indirectly.

In practice, microtiter plates can conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, can be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for target geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

5.8.2. ASSAYS FOR CELLULAR PROTEINS THAT INTERACT WITH THE TARGET GENEPROTEIN

Any method suitable for detecting protein-protein interactions can beemployed for identifying novel target protein-cellular or extracellularprotein interactions. These methods are outlined in Section 5.2., above,for the identification of pathway genes, and can be utilized herein withrespect to the identification of proteins which interact with identifiedtarget proteins.

5.8.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH TARGET GENEPRODUCT/CELLULAR MACROMOLECULE INTERACTION

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.Such macromolecules can include, but are not limited to, nucleic acidmolecules and those proteins identified via methods such as thosedescribed, above, in Section 5.8.2. For purposes of this discussion,such cellular and extracellular macromolecules are referred to herein as"binding partners". Compounds that disrupt such interactions can beuseful in regulating the activity of the target gene protein, especiallymutant target gene proteins. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and the like, asdescribed, for example, in Section 5.8.1. above.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the target gene product and itscellular or extracellular binding partner or partners involves preparinga reaction mixture containing the target gene product and the bindingpartner under conditions and for a time sufficient to allow the two tointeract and bind, thus form a complex. In order to test a compound forinhibitory activity, the reaction mixture is prepared in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of target gene product and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene protein and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene protein and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene protein can also be compared to complex formationwithin reaction mixtures containing the test compound and a mutanttarget gene protein. This comparison can be important in those caseswherein it is desirable to identify compounds that disrupt interactionsof mutant but not normal target gene proteins.

The assay for compounds that interfere with the interaction of thetarget gene products and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the target gene product or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the target gene products and the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thetarget gene protein and interactive cellular or extracellular bindingpartner. Alternatively, test compounds that disrupt preformed complexes,e.g. compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the target gene protein or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtiter plates are convenientlyutilized. The anchored species can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished simplyby coating the solid surface with a solution of the target gene productor binding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored can be used to anchor thespecies to the solid surface. The surfaces can be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the target gene proteinand the interactive cellular or extracellular binding partner isprepared in which either the target gene product or its binding partneris labeled, but the signal generated by the label is quenched due tocomplex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubensteinwhich utilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances which disrupt target geneprotein/cellular or extracellular binding partner interaction can beidentified.

In a particular embodiment, the target gene product can be prepared forimmobilization using recombinant DNA techniques described in Section5.5, above. For example, the target gene coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive cellular or extracellularbinding partner can be purified and used to raise a monoclonal antibody,using methods routinely practiced in the art and described above, inSection 5.6. This antibody can be labeled with the radioactive isotope¹²⁵ I, for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-target gene fusion protein can beanchored to glutathione-agarose beads. The interactive cellular orextracellular binding partner can then be added in the presence orabsence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed components. Theinteraction between the target gene protein and the interactive cellularor extracellular binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-target gene fusion protein and the interactivecellular or extracellular binding partner can be mixed together inliquid in the absence of the solid glutathione-agarose beads. The testcompound can be added either during or after the species are allowed tointeract. This mixture can then be added to the glutathione-agarosebeads and unbound material is washed away. Again the extent ofinhibition of the target gene product/binding partner interaction can bedetected by adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the target gene product and/or the interactive cellular orextracellular binding partner (in cases where the binding partner is aprotein), in place of one or both of the full length proteins. Anynumber of methods routinely practiced in the art can be used to identifyand isolate the binding sites. These methods include, but are notlimited to, mutagenesis of the gene encoding one of the proteins andscreening for disruption of binding in a co-immunoprecipitation assay.Compensating mutations in the gene encoding the second species in thecomplex can then be selected. Sequence analysis of the genes encodingthe respective proteins will reveal the mutations that correspond to theregion of the protein involved in interactive binding. Alternatively,one protein can be anchored to a solid surface using methods describedin this Section above, and allowed to interact with and bind to itslabeled binding partner, which has been treated with a proteolyticenzyme, such as trypsin. After washing, a short, labeled peptidecomprising the binding domain can remain associated with the solidmaterial, which can be isolated and identified by amino acid sequencing.Also, once the gene coding for the cellular or extracellular bindingpartner is obtained, short gene segments can be engineered to expresspeptide fragments of the protein, which can then be tested for bindingactivity and purified or synthesized.

For example, and not by way of limitation, a target gene product can beanchored to a solid material as described, above, in this Section, bymaking a GST-target gene fusion protein and allowing it to bind toglutathione agarose beads. The interactive cellular or extracellularbinding partner can be labeled with a radioactive isotope, such as ³⁵ S,and cleaved with a proteolytic enzyme such as trypsin. Cleavage productscan then be added to the anchored GST-target gene fusion protein andallowed to bind. After washing away unbound peptides, labeled boundmaterial, representing the cellular or extracellular binding partnerbinding domain, can be eluted, purified, and analyzed for amino acidsequence by well known methods. Peptides so identified can be producedsynthetically or fused to appropriate facilitative proteins using wellknown recombinant DNA technology.

5.8.4 ASSAYS FOR AMELIORATION OF IMMUNE DISORDER SYMPTOMS AND/OR THEMODULATION OF TARGET GENE PRODUCT FUNCTION

Any of the binding compounds, including but not limited to, compoundssuch as those identified in the foregoing assay systems, can be testedfor the ability to ameliorate symptoms of immune disorders e.g., TH cellsubpopulation-related disorders. Cell-based and animal model-basedassays for the identification of compounds exhibiting such an ability toameliorate immune disorder symptoms are described below. Further,cell-based assays for the identification of compounds which modulatetarget gene product function, in instances where the target gene productis a receptor having a seven transmembrane domain sequence, such as, forexample, that of the 10 gene product, are described, below, in Section5.8.4.1.

First, cell-based systems such as those described, above, in Section5.7.2, can be used to identify compounds which can act to ameliorate THcell subpopulation-related disorder symptoms. For example, such cellsystems can be exposed to a compound, suspected of exhibiting an abilityto ameliorate the disorder symptoms, at a sufficient concentration andfor a time sufficient to elicit such an amelioration in the exposedcells. After exposure, the cells are examined to determine whether oneor more of the TH cell subpopulation-related disorder-like cellularphenotypes has been altered to resemble a phenotype more likely toproduce a lower incidence or severity of disorder symptoms. Additionalcell-based assays are discussed, below, in Section 5.8.4.1.

Taking the TH cell subpopulation-related disorder asthma, which is,specifically, a TH2-like-related disorder, any TH2 or TH2-like cellsystem can be utilized. Upon exposure to such cell systems, compoundscan be assayed for their ability to modulate the TH2-like phenotype ofsuch cells, such that the cells exhibit loss of a TH2-like phenotype.Compounds with such TH2 modulatory capability represent ones which canpotentially exhibit the ability to ameliorate asthma-related symptoms invivo.

In addition, animal-based systems, such as those described, above, inSection 5.7.1, can be used to identify compounds capable of amelioratingTH cell subpopulation-related disorder-like symptoms. Such animal modelscan be used as test substrates for the identification of drugs,pharmaceuticals, therapies, and interventions which can be effective intreating such disorders. For example, animal models can be exposed to acompound, suspected of exhibiting an ability to ameliorate TH cellsubpopulation-related disorder symptoms, at a sufficient concentrationand for a time sufficient to elicit such an amelioration of the symptomsin the exposed animals. The response of the animals to the exposure, andthus the efficacy of the compound in question, can be monitored byassessing the reversal of disorders associated with TH cellsubpopulation-related disorders of interest. With regard tointervention, any treatments which reverse any aspect of TH cellsubpopulation-related disorder-like symptoms should be considered ascandidates for corresponding human TH cell subpopulation-relateddisorder therapeutic intervention. Dosages of test agents can bedetermined by deriving dose-response curves, as discussed in Section5.10, below.

Gene expression patterns can be utilized in conjunction with eithercell-based or animal-based systems, to assess the ability of a compoundto ameliorate TH cell subpopulation-related disorder-like symptoms. Forexample, the expression pattern of one or more fingerprint genes canform part of a fingerprint profile which can be then be used in such anassessment. Fingerprint profiles are described, below, in Section 5.11.Fingerprint profiles can be characterized for known states, either THcell subpopulation-related disorder states, or normal TH celldifferentiative states, within the cell- and/or animal-based modelsystems.

5.8.4.1. METHODS FOR THE IDENTIFICATION OF COMPOUNDS WHICH MODULATETARGET GENE PRODUCT FUNCTION

In this Section, methods are described for the identification ofcompounds which act as either agonists or antagonists of receptor targetgene products. The 10 gene product (FIG. 9; SEQ ID NO:9) is an exampleof a seven transmembrane-domain target gene product. For ease ofexplanation, and not by way of limitation, therefore, the 10 geneproduct will be used to illustrate the methods described in thisSection.

The compounds tested may be, for example, compounds such as thoseidentified via the assays described, above, in Sections 5.8.1 to 5.8.3.Such compounds may include, but are not limited to peptides such as, forexample, soluble peptides, including, but not limited to, Ig-tailedfusion peptides, comprising extracellular portions of target geneproduct transmembrane receptors, and members of random peptide libraries(see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghten, R. etal., 1991, Nature 354:84-86) made of D-and/or L-configuration aminoacids, phosphopeptides (including but not limited to members of randomor partially degenerate, directed phosphopeptide libraries; see, e.g.,Songyang, Z. et al., 1993, Cell 72:767-778), antibodies (including, butnot limited to polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expressionlibrary fragments, and epitope-binding fragments thereof), and smallorganic or inorganic molecules.

The assays described herein are functional assays which identifycompounds that affect the receptor target gene's activity by affectingthe level of intracellular calcium release within cells expressing suchseven transmembrane domain receptor target protein (e.g., the 10 geneproduct). Intracellular calcium release is measured because such seventransmembrane domain receptors tend to be G protein-coupled receptorsand because activation of these receptors leads to a G protein-mediatedintracellular calcium release. Modulation (i.e., agonization orantagonization) of the receptor target gene product function, then,would result in a difference in intracellular calcium levels.

The assays comprise contacting a seven transmembrane domain receptortarget gene-expressing cell with a test compound and measuring the levelof intracellular calcium. Those compounds which produce an intracellularcalcium profile which differs from that which the cell would exhibit inthe absence of the compound represent either agonists or antagonists. Anagonist compound would cause an increase in intracellular calcium levelsrelative to control cells while an antagonist would result in a decreasein intracellular calcium levels relative to control cells.

While any cell expressing a seven transmembrane receptor target geneproduct may be used herein, it is preferred that cells be used whoseintracellular calcium levels may readily measured. Xenopus oocytes, dueto their large size, are among such preferred cells because they caneasily be injected with intracellular calcium reporter compounds.Additionally, myeloma cells may be utilized. Such reporter compoundsinclude, but are not limited to, calcium-binding agents such as the wellknown FURA-2 and INDO-2. FURA-2/calcium complexes and INDO-2/calciumcomplexes fluoresce, making possible the measurement of differences inintracellular calcium levels.

For the purposes of the assays described herein, the Xenopus oocytesshould be transfected with nucleotide sequences encoding the targetprotein of interest (e.g., the 10 gene product). The cells can betransfected and express the sequence of interest via techniques whichare well known to those of skill in the art and which may include, forexample, techniques such as those described, above, in Section 5.5.Xenopus oocytes can be injected with RNA encoding the target geneproduct of interest such that the injected oocytes will express the geneproduct.

The assays described in this Section may, first, be used to identifycompounds which act as agonists of the target gene product of interest,e.g., the 10 gene product. "Agonist", as used herein, refers to acompound which modulates target gene product activity by increasing thetarget gene product's activity, as evaluated by the compound's abilityto bring about an increase in calcium influx, leading to an increaseintracellular calcium levels.

Among such agonists can be, for example, the natural ligand for thereceptor target gene product, e.g., the natural ligand for the 10 geneproduct.

Agonists identified via such assays may act as useful therapeutic agentsfor the amelioration of a wide range of T cell-related disorders,including, for example, TH cell subpopulation-related disorders, ininstances whereby such disorders are caused by a reduced or absent levelof target gene product activity. Any of the agonist compounds identifiedherein can be used, for example, as part of the treatment methodsdescribed in Section 5.9.2, below. Further, such agonists can be used toidentify antagonists of the receptor target gene product of interest,e.g., as described, below.

"Antagonist", as used herein, refers to a compound which modulatestarget gene product activity by decreasing the target gene product'sactivity, as evaluated by the compound's ability to bring about adecrease in calcium influx. Antagonists identified via such assays mayact as useful therapeutic agents for the amelioration of a wide range ofT cell-related disorders, including, for example, TH cellsubpopulation-related disorders, in instances whereby the disorder iscaused by an increased or inappropriate level of target gene productactivity.

An antagonist screen may be performed utilizing target geneproduct-expressing cells as described, above, and which include, but arenot limited to, such cells as 10 gene-expressing cells, for example, 10gene-expressing Xenopus oocytes. In those instances whereby the Tcell-related disorder is caused by a mutant target gene product, thecells utilized in the antagonist assay can be cells which express themutant receptor target gene product involved in causing the Tcell-related disorder.

To conduct an antagonist screen, a target gene-expressing cell iscontacted with 1) an agonist of the target gene product and 2) a testcompound for a given period of time. The level of intracellular calciumis then measured in the cells and in cells which have been contactedwith agonist alone. A test compound is considered to be an antagonist ifthe level of intracellular calcium release in the presence of the testcompound is lower than the level of intracellular calcium release in theabsence of the test compound.

Any of the antagonist compounds identified herein can be used, forexample, as part of the treatment methods described, below, in Section5.9.1.

Among the potential antagonist compounds of the seven transmembranedomain receptor target gene products described herein are peptides whichcontain one or more of the receptor target gene product's extracellulardomains, preferably those domains are domains which are responsible forligand-binding such that the peptides act to compete with the endogenousreceptor for ligand. In the case of the 10 gene product, for example,such extracellular domains include from approximately 10 gene productamino acid residue 1 to 19, amino acid residue 74 to 87, amino acidresidue 153-187 and amino acid residue 254 to 272. Such extracellulardomain antagonist compounds may comprise soluble Ig-tailed fusionproteins which may be produced by utilizing techniques such as thosedescribed, above, in Section 5.5. Additionally, antibodies directedagainst the extracellular portion of the 10 gene product may reduce 10gene product function by, for example, blocking ligand binding.

5.9. Compounds and Methods for Treatment of Immune Disorders and forModulation of TH Cell Responsiveness

Described below are methods and compositions which can be used toameliorate immune disorder symptoms via, for example, a modulation ofthe TH cell subpopulation of interest. Such modulation can be of apositive or negative nature, depending on the specific situationinvolved, but each modulatory event yields a net result in whichsymptoms of the immune disorder are ameliorated. Further, describedbelow are methods for the modulation of TH cell responsiveness toantigen.

"Negative modulation", as used herein, refers to a reduction in thelevel and/or activity of target gene product relative to the leveland/or activity of the target gene product in the absence of themodulatory treatment. Alternatively, the term, as used herein, refers toa depletion of the T cell subpopulation (e.g., via a reduction in thenumber of cells belonging to the TH cell subpopulation) relative to thenumber present in the absence of the modulatory treatment. "Depletion,"as used herein, is as defined, above, in Section 3.

"Positive modulation", as used herein, refers to an increase in thelevel and/or activity of target gene product relative to the leveland/or activity of the gene product in the absence of the modulatorytreatment. Alternatively, the term, as used herein, refers to astimulation of the T cell subpopulation (e.g., via an increase in thenumber of cells belonging to the TH cell subpopulation), relative to thenumber present in the absence of the modulatory treatment."Stimulation," as used herein, is as defined, above, in Section 3.

It is possible that a TH cell subpopulation-related disorder or otherimmune disorder, can occur as a result of normal target gene activityduring the course of, for example, exposure to a certain antigen whichelicits an immune response that leads to the development of thedisorder. For example, the TH2-like-related disorders, asthma andallergy, are likely candidates of disorders having such a mechanism.Additionally, a disorder can be brought about, at least in part, by anabnormally high level of target gene product, or by the presence of atarget gene product exhibiting an abnormal activity. As such, atechnique which elicits a negative modulatory effect, i.e., brings abouta reduction in the level and/or activity of target gene product, oralternatively, brings about a depletion of the TH cell subpopulation(e.g., via a physical reduction in the number of cells belonging to theTH cell subpopulation), would effect an amelioration of TH cellsubpopulation-related disorder symptoms in either of the abovescenarios.

Negative modulatory techniques for the reduction of target geneexpression levels or target gene product activity levels, (either normalor abnormal), and for the reduction in the number of specific TH cellsubpopulation cells are discussed in Section 5.9.1, below.

Alternatively, it is possible that a TH cell subpopulation-relateddisorder or other immune disorders can be brought about, at least inpart, by the absence or reduction of the level of target geneexpression, a reduction in the level of a target gene product'sactivity, or a reduction in the overall number of cells belonging to aspecific TH cell subpopulation. As such, a technique which elicits apositive modulatory effect, i.e., brings about an increase in the levelof target gene expression and/or the activity of such gene products, or,alternatively, a stimulation of the TH cell subpopulation (e.g., via aphysical increase in the number of cells belonging to a TH cellsubpopulation), would effect an amelioration of immune disordersymptoms.

For example, a reduction in the overall number of TH1-like cellsrelative to TH2-like cells within a HIV-infected individual cancorrelate with the progression to AIDS (Clerci, M. et al., 1993, J.Clin. Invest. 91:759; Clerci, M. et al., 1993, Science 262:1721; Maggi,E. et al., 1994, Science 265:244). A treatment capable of increasing thenumber of TH1-like cells relative to TH2-like cells within anHIV-infected individual may, therefore, serve to prevent or slow theprogression to disease.

Positive modulatory techniques for increasing target gene expressionlevels or target gene product activity levels, and for increasing thelevel of specific TH cell subpopulation cells are discussed, below, inSection 5.9.2.

Among the immune disorders whose symptoms can be ameliorated are TH1 orTH1-like related immune disorders and TH2 or TH2-like related immunedisorders. Examples of TH1 or TH1-like related disorders include chronicinflammatory diseases and disorders, such as Crohn's disease, reactivearthritis, including Lyme disease, insulin-dependent diabetes,organ-specific autoimmunity, including multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease, contact dermatitis, psoriasis, graftrejection, graft versus host disease and sarcoidosis. Examples of TH2 orTH2-like related disorders include atopic conditions, such as asthma andallergy, including allergic rhinitis, gastrointestinal allergies,including food allergies, eosinophilia, conjunctivitis, glomerularnephritis, certain pathogen susceptibilities such as helminthic (e.g.,leishmaniasis) and certain viral infections, including HIV, andbacterial infections, including tuberculosis and lepromatous leprosy.

The methods described herein can additionally be utilized the modulatethe level of responsiveness, for example, responsiveness to antigen, ofa TH cell subpopulation. Such methods are important in that many immunedisorders involve inappropriate rather than insufficient immuneresponses. For example, disorders such as atopic, IgE-mediated allergicconditions, including asthma, pathogen susceptibilities and chronicinflammatory disease, involve strong but counterproductive TH2-mediatedimmune responses. Further, inappropriate TH1-mediated immune responsesto self-antigens is central to the development of such disorders asmultiple sclerosis, psoriasis, insulin dependent diabetes, Hashimoto'sthyroiditis and Crohn's disease.

Methods for modulating TH cell responsiveness can comprise, for example,contacting a compound to a TH cell so that the responsiveness of the Thelper cell is modulated relative to the responsiveness of the T helpercell in the absence of the compound. The modulation can increase ordecrease the responsiveness of the TH cell. Any of the techniquesdescribed, below, in Sections 5.9.1-5.9.3.2 can be utilized to effect anappropriate modulation of TH cell responsiveness.

5.9.1 Negative Modulatory Techniques

As discussed, above, successful treatment of certain immune disorderscan be brought about by techniques which serve to inhibit the expressionor activity of target gene products, or which, alternatively, serve toreduce the overall number of cells belonging to a specific TH cellsubpopulation.

For example, compounds such as those identified through assaysdescribed, above, in Section 5.8, which exhibit negative modulatoryactivity, can be used in accordance with the invention to amelioratecertain TH cell subpopulation-related disorder symptoms. As discussed inSection 5.8, above, such molecules can include, but are not limited topeptides (such as, for example, peptides representing solubleextracellular portions of target gene product transmembrane receptors),phosphopeptides, small organic or inorganic molecules, or antibodies(including, for example, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂and FAb expression library fragments, and epitope-binding fragmentsthereof). Techniques for the determination of effective doses andadministration of such compounds are described, below, in Section 5.10.

Further, antisense and ribozyme molecules which inhibit expression ofthe target gene can also be used in accordance with the invention toreduce the level of target gene expression, thus effectively reducingthe level of target gene activity. Still further, triple helix moleculescan be utilized in reducing the level of target gene activity. Suchtechniques are described, below, in Section 5.9.1.1.

Additionally, techniques for the depletion of specific TH cellsubpopulations are discussed, below, in Section 5.9.3. Such techniquescan take advantage of, for example, novel cell surface markers which arespecific to the TH cell subpopulation to be depleted, and can include invivo or in vitro targeted destruction, or, alternatively, selectivepurification away, of the TH cell subpopulation of interest.

Among the TH cell subpopulation-related sequences identified by themethods described by the present invention is a gene designated hereinas the 103 gene, as discussed in the Example presented in Section 7,below. The 103 gene is demonstrated herein to represent a TH2-specificgene in that 103 gene expression is found to be absent TH1 cells as wellas all other tissues tested. Further, at least one of the proteinsproduced by the 103 gene is a transmembrane protein.

The 103 gene and its products can, therefore, be utilized in thetreatment of TH2 cell subpopulation-related disorders. For example, a103 gene product or portions thereof can be utilized, either directly orindirectly, to ameliorate conditions involving inappropriate IgE immuneresponses, including, but not limited to the symptoms which accompanyatopic conditions such as allergy and/or asthma. IgE-type antibodies areproduced by stimulated B cells which require, at least in part, IL-4produced by the TH2 cell subpopulation. Therefore, any treatment,including, for example, the use of a gene 103 product or portionthereof, which reduces the effective concentration of secreted IL-4,e.g., by reducing the number or activity of TH2 cells, can bring about areduction in the level of circulating IgE, leading, in turn, to theamelioration of the conditions stemming from an inappropriate IgE immuneresponse.

There exist a variety of ways in which the TH2 specific 103 geneproducts can be used to effect such a reduction in the activity and/oreffective concentration of TH2 cells. For example, natural ligands,derivatives of natural ligands and antibodies which bind to the 103 geneproduct can be utilized to reduce the number of TH2 cells present byeither physically separating such cells away from other cells in apopulation, thereby deleting the TH2 cell subpopulation, or,alternatively, by targeting the specific destruction of TH2 cells. Suchtechniques are discussed, below, in Section 5.9.3. Further, suchcompounds can be used to inhibit the proliferation of TH2 cells.

Additionally, compounds such as 103 gene sequences or gene products canbe utilized to reduce the level of TH2 cell activity, cause a reductionin IL-4 production, and, ultimately, bring about the amelioration of IgErelated disorders.

For example, compounds can be administered which compete with endogenousligand for the 103 gene product. The resulting reduction in the amountof ligand-bound 103 gene transmembrane protein will modulate TH2cellular activity. Compounds which can be particularly useful for thispurpose include, for example, soluble proteins or peptides, such aspeptides comprising the extracellular domain, or portions and/or analogsthereof, of the gene 103 product, including, for example, soluble fusionproteins such as Ig-tailed fusion proteins. (For a discussion of theproduction of Ig-tailed fusion proteins see, for example, U.S. PatentNo. 5,116,964.)

The novel 200 gene, which encodes a receptor target gene product that isa member of the Ig superfamily, exhibits a TH1-specific pattern of geneexpression. The 200 gene and its products can, therefore, be utilized inthe treatment of TH1 cell subpopulation-related disorders such as, forexample, chronic inflammatory diseases, psoriasis, graft rejection andgraft versus host disease.

The treatment of such disorder may require a reduction in the activityand/or effective concentration of the TH1 cell subpopulation involved inthe disorder of interest. As such, a number of methods exist whereby theTH1 specific 200 gene products can be used to effect such a reduction inthe activity and/or effective concentration of TH1 cells. For example,natural ligands, derivatives of natural ligands and antibodies whichbind to the 200 gene product can be utilized to reduce the number of TH1cells present by either physically separating such cells away from othercells in a population, thereby deleting the TH1 cell subpopulation, or,alternatively, by targeting the specific destruction of TH1 cells. Suchtechniques are discussed, below, in Section 5.9.3. Further, suchcompounds can be used to inhibit the proliferation of TH1 cells.

Additionally, compounds can be administered which compete withendogenous ligand for the 200 gene product. The resulting reduction inthe amount of ligand-bound 200 gene transmembrane protein will modulateTH1 cellular activity. Compounds which can be particularly useful forthis purpose include, for example, soluble proteins or peptides, such aspeptides comprising the extracellular domain, or portions and/or analogsthereof, of the gene 200 product, including, for example, soluble fusionproteins such as Ig-tailed fusion proteins. (For a discussion of theproduction of Ig-tailed fusion proteins see, for example, U.S. Pat. No.5,116,964.)

The 10 gene product is identified herein as a receptor target geneproduct having a seven transmembrane domain sequence motif. Further, the10 gene is shown to exhibit a TH inducible pattern of expression,meaning that 10 gene expression increases in both TH1 and TH2 cellsubpopulations in response to stimulation and can important to T cellresponses in general. The 10 gene and its products can, therefore, beutilized in the treatment of a wide T cell-related disorders. Techniquessuch as those described, above, for the 103 and the 200 genes and geneproducts can also be utilized for the amelioration of disorders in which10 gene expression is involved.

5.9.1.1. Negative Modulatory Antisense, Ribozyme and Triple HelixApproaches

Among the compounds which can exhibit the ability to ameliorate TH cellsubpopulation-related disorder symptoms are antisense, ribozyme, andtriple helix molecules. Such molecules can be designed to reduce orinhibit either wild type, or if appropriate, mutant target geneactivity. Techniques for the production and use of such molecules arewell known to those of skill in the art.

Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by hybridizing to targeted mRNA and preventing proteintranslation. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the -10 and+10 regions of the target gene nucleotide sequence of interest, arepreferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA (For a review see, for example Rossi, J., 1994, CurrentBiology 4:469-471). The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by a endonucleolytic cleavage. The composition of ribozymemolecules must include one or more sequences complementary to the targetgene mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see U.S. Pat. No.5,093,246, which is incorporated by reference herein in its entirety. Assuch, within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of RNA sequences encoding target gene proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the molecule of interest for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable. The suitability of candidatesequences can also be evaluated by testing their accessibility tohybridize with complementary oligonucleotides, using ribonucleaseprotection assays.

Nucleic acid molecules to be used in triplex helix formation for theinhibition of transcription should be single stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides must bedesigned to promote triple helix formation via Hoogsteen base pairingrules, which generally require sizeable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences can be pyrimidine-based, which will result in TAT and CGC⁺triplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules can bechosen that are purine-rich, for example, contain a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation can be increased by creating a so-called "switchback"nucleic acid molecule. Switchback molecules are synthesized in analternating 5'-3', 3'-5' manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In instances wherein the antisense, ribozyme, and/or triple helixmolecules described herein are utilized to inhibit mutant geneexpression, it is possible that the technique can also efficientlyreduce or inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene allelesthat the possibility can arise wherein the concentration of normaltarget gene product present can be lower than is necessary for a normalphenotype. In such cases, to ensure that substantially normal levels oftarget gene activity are maintained, therefore, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethods such as those described, below, in Section 5.9.2 that do notcontain sequences susceptible to whatever antisense, ribozyme, or triplehelix treatments are being utilized. Alternatively, in instances wherebythe target gene encodes an extracellular protein, it can be preferableto coadminister normal target gene protein in order to maintain therequisite level of target gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of themolecule or the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

5.9.2. Positive Modulatory Techniques

As discussed above, successful treatment of certain immune disorders canbe brought about by techniques which serve to increase the level oftarget gene expression or to increase the activity of target geneproduct, or which, or alternatively, serve to effectively increase theoverall number of cells belonging to a specific TH cell subpopulation.

For example, compounds such as those identified through assaysdescribed, above, in Section 5.8, which exhibit positive modulatoryactivity can be used in accordance with the invention to amelioratecertain TH cell subpopulation-related disorder symptoms. As discussed inSection 5.8, above, such molecules can include, but are not limited topeptides representing soluble extracellular portions of target geneproduct transmembrane proteins, phosphopeptides, small organic orinorganic molecules, or antibodies (including, for example, polyclonal,monoclonal, humanized, anti-idiotypic, chimeric or single chainantibodies, and FAb, F(ab')₂ and FAb expression library fragments, andepitope-binding fragments thereof).

For example, a compound, such as a target gene protein, can, at a levelsufficient to ameliorate immune disorder symptoms, be administered to apatient exhibiting such symptoms. Any of the techniques discussed,below, in Section 5.10, can be utilized for such administration. One ofskill in the art will readily know how to determine the concentration ofeffective, non-toxic doses of the compound, utilizing techniques such asthose described, below, in Section 5.10.1.

In instances wherein the compound to be administered is a peptidecompound, DNA sequences encoding the peptide compound can be directlyadministered to a patient exhibiting immune disorder symptoms, at aconcentration sufficient to produce a level of peptide compoundsufficient to ameliorate the disorder symptoms. Any of the techniquesdiscussed, below, in Section 5.10, which achieve intracellularadministration of compounds, such as, for example, liposomeadministration, can be utilized for the administration of such DNAmolecules. The DNA molecules can be produced, for example, by well knownrecombinant techniques.

In the case of peptides compounds which act extracellularly, the DNAmolecules encoding such peptides can be taken up and expressed by anycell type, so long as a sufficient circulating concentration of peptideresults for the elicitation of a reduction in the immune disordersymptoms. In the case of compounds which act intracellularly, the DNAmolecules encoding such peptides must be taken up and expressed by theTH cell subpopulation of interest at a sufficient level to bring aboutthe reduction of immune disorders.

Any technique which serves to selectively administer DNA molecules tothe TH cell subpopulation of interest is, therefore, preferred, for theDNA molecules encoding intracellularly acting peptides. In the case ofasthma, for example, techniques for the selective administration of themolecules to TH cell subpopulations residing within lung tissue arepreferred.

Further, in instances wherein the TH cell subpopulation-related disorderinvolves an aberrant gene, patients can be treated by gene replacementtherapy. One or more copies of a normal target gene or a portion of thegene that directs the production of a normal target gene protein withtarget gene function, can be inserted into cells, using vectors whichinclude, but are not limited to adenovirus, adeno-associated virus, andretrovirus vectors, in addition to other particles that introduce DNAinto cells, such as liposomes.

Such gene replacement techniques can be accomplished either in vivo orin vitro. As above, for genes encoding extracellular molecules, the celltype expressing the target gene is less important than achieving asufficient circulating concentration of the extracellular molecule forthe amelioration of immune disorders. Further, as above, when the geneencodes a cell which acts intracellularly or as a transmembranemolecule, the gene must be expressed with the TH cell subpopulation celltype of interest. Techniques which select for expression within the celltype of interest are, therefore, preferred for this latter class oftarget genes. In vivo, such techniques can, for example, includeappropriate local administration of target gene sequences.

In vitro, target gene sequences can be introduced into autologous cells.These cells expressing the target gene sequence of interest can then bereintroduced, preferably by intravenous administration, into the patientsuch that there results an amelioration of the symptoms of the disorder.

Alternatively, TH cells belonging to a specific TH cell subpopulationcan be administered to a patient such that the overall number of cellsbelonging to that TH cell subpopulation relative to other TH cellsubpopulation cells is increased, which results in an amelioration of aTH cell subpopulation-related disorder. Techniques for such TH cellsubpopulation augmentation are described, below, in Section 5.9.

5.9.3 Negative or Positive Modulatory Techniques

Described herein are modulatory techniques which, depending on thespecific application for which they are utilized, can yield eitherpositive or negative responses leading to the amelioration of immunedisorders, including TH cell subpopulation-related disorders. Thus, inappropriate instances, the procedures of this Section can be used inconjunction with the negative modulatory techniques described, above, inSection 5.9.1 or, alternatively, in conjunction with the positivemodulatory techniques described, above, in Section 5.9.2.

5.9.3.1. Antibody Techniques

Antibodies exhibiting modulatory capability can be utilized toameliorate immune disorders such as TH cell subpopulation-relateddisorders. Depending on the specific antibody, the modulatory effect canbe negative and can, therefore, by utilized as part of the techniquesdescribed, above, in Section 5.9.1, or can be positive, and can,therefore, be used in conjunction with the techniques described, above,in Section 5.9.2.

An antibody having negative modulatory capability refers to an antibodywhich specifically binds to and interferes with the action of a protein.In the case of an extracellular receptor, for example, such an antibodywould specifically bind the extracellular domain of the receptor in amanner which does not activate the receptor but which disrupts theability of the receptor to bind its natural ligand. For example,antibodies directed against the extracellular domains of genes 103 or200 can function as such negative modulators. Additionally, antibodiesdirected against one or more of the 10 gene product extracellulardomains can function in a negative modulatory manner. Such antibodiescan be generated using standard techniques described in Section 5.6,above, against full length wild type or mutant proteins, or againstpeptides corresponding to portions of the proteins. The antibodiesinclude but are not limited to polyclonal, monoclonal, FAb fragments,single chain antibodies, chimeric antibodies, and the like.

An antibody having positive modulatory capability refers to an antibodywhich specifically binds to a protein and, by binding, serves to, eitherdirectly or indirectly, activate the function of the protein which itrecognizes. For example, an antibody can bind to the extracellularportion of a transmembrane protein in a manner which causes thetransmembrane protein to function as though its endogenous ligand wasbinding, thus activating, for example, a signal transduction pathway.Antibodies can be generated using standard techniques described inSection 5.6, above, against full length wild type or mutant proteins, oragainst peptides corresponding to portions of the proteins. Theantibodies include but are not limited to polyclonal, monoclonal, FAbfragments, single chain antibodies, chimeric antibodies, and the like.

In instances where the protein, such as a target gene protein, to whichthe antibody is directed is intracellular and whole antibodies are used,internalizing antibodies can be preferred. However, lipofectin orliposomes can be used to deliver the antibody or a fragment of the Fabregion which binds to the gene product epitope into cells. Wherefragments of the antibody are used, the smallest inhibitory fragmentwhich binds to the protein's binding domain is preferred. For example,peptides having an amino acid sequence corresponding to the domain ofthe variable region of the antibody that binds to the protein can beused. Such peptides can be synthesized chemically or produced viarecombinant DNA technology using methods well known in the art (e.g.,see Creighton, 1983, supra; and Sambrook et al., 1989, above).Alternatively, single chain antibodies, such as neutralizing antibodies,which bind to intracellular epitopes can also be administered. Suchsingle chain antibodies can be administered, for example, by expressingnucleotide sequences encoding single-chain antibodies within the targetcell population by utilizing, for example, techniques such as thosedescribed in Marasco et al. (Marasco, W. et al., 1993, Proc. Natl. Acad.Sci. USA 90:7889-7893).

In instances where the protein to which the antibody is directed isextracellular, or is a transmembrane protein, any of the administrationtechniques described, below in Section 5.10 which are appropriate forpeptide administration can be utilized to effectively administer theantibodies to their site of action.

5.9.3.2 Methods for Increasing or Decreasing Specific TH CellSubpopulation Concentrations

Techniques described herein can be utilized to either deplete or augmentthe total number of cells belonging to a given TH cell subpopulation,thus effectively increasing or decreasing the ratio of the TH cellsubpopulation of interest to other TH cell subpopulations. Specifically,separation techniques are described which can be used to either depleteor augment the total number of cells present within a TH cellsubpopulation, and, further, targeting techniques are described whichcan be utilized to deplete specific TH cell subpopulations.

Depending on the particular application, changing the number of cellsbelonging to a TH cell subpopulation can yield either stimulatory orinhibitory responses leading to the amelioration of TH cellsubpopulation disorders. Thus, in appropriate instances, the proceduresof this Section can be used in conjunction with the inhibitorytechniques described, above, in Section 5.9.1. or, alternatively, inconjunction with the stimulatory techniques described, above, in Section5.9.2.

The separation techniques described herein are based on the presence orabsence of specific cell surface markers, preferably transmembranemarkers. Such markers can include, but are not limited to, theTH2-specific 103 gene product extracellular domain markers, theTH1-specific 200 gene product extracellular domain markers and the THinducible 10 gene product extracellular domain markers.

In instances wherein the goal of the separation is to increase oraugment the number of cells belonging to a specific TH cellsubpopulation, the antibodies used can also be specific to surfacemarkers present on undifferentiated or partially undifferentiated THcells. After separation, and purification of such undifferentiated orpartially differentiated TH cells, the cells can be cultured inphysiological buffer or culture medium and induced to differentiate byculturing in the presence of appropriate factors. For example, IL-4 canbe added to induce the TH cells to differentiate into TH2 cells, whilethe cytokine IL-12 can be added to induce the TH cells to differentiateinto TH1 cells. After differentiation, cells can be washed, resuspendedin, for example, buffered saline, and reintroduced into a patient via,preferably, intravenous administration.

Separation techniques can be utilized which separate and purify cells,in vitro, from a population of cells, such as hematopoietic cellsautologous to the patient being treated. An initial TH cellsubpopulation-containing population of cells, such as hematopoieticcells, can be obtained using standard procedures well known to those ofskill in the art. Peripheral blood can be utilized as one potentialstarting source for such techniques, and can, for example, be obtainedvia venipuncture and collection into heparinized tubes.

Once the starting source of autologous cells is obtained, the T cells,such as TH1 or TH2 cells, can be removed, and thus selectively separatedand purified, by various methods which utilize antibodies which bindspecific markers present on the T cell population of interest, whileabsent on other cells within the starting source. These techniques caninclude, for example, flow cytometry using a fluorescence activated cellsorter (FACS) and specific fluorochromes, biotin-avidin orbiotin-streptavidin separations using biotin conjugated to cell surfacemarker-specific antibodies and avidin or streptavidin bound to a solidsupport such as affinity column matrix or plastic surfaces or magneticseparations using antibody-coated magnetic beads.

Separation via antibodies for specific markers can be by negative orpositive selection procedures. In negative separation, antibodies areused which are specific for markers present on undesired cells. Forexample, in the case of a TH1 cell subpopulation-related disorderwherein it would be desirable to deplete the number of TH1 cells, suchantibodies could be directed to the extracellular domain of the 200 geneproduct. Alternatively, in the case of TH2 cell subpopulation-relateddisorders wherein it would be desirable to deplete the number of TH1cells, such antibodies could be directed to the extracellular domain ofthe 103 gene product. Cells bound by an antibody to such a cell surfacemarker can be removed or lysed and the remaining desired mixtureretained.

In positive separation, antibodies specific for markers present on thedesired cells of interest. For example, in the case of a TH1 cellsubpopulation-related disorder wherein it would be desirable to increasethe number of TH1 cells, such antibodies could be directed to theextracellular domain of the 200 gene product. Alternatively, in the caseof TH2 cell subpopulation-related disorders wherein it would bedesirable to increase the number of TH1 cells, such antibodies could bedirected to the extracellular domain of the 103 gene product. Cellsbound by the antibody are separated and retained. It will be understoodthat positive and negative separations can be used substantiallysimultaneously or in a sequential manner.

A common technique for antibody based separation is the use of flowcytometry such as by a florescence activated cell sorter (FACS).Typically, separation by flow cytometry is performed as follows. Thesuspended mixture of cells are centrifuged and resuspended in media.Antibodies which are conjugated to fluorochrome are added to allow thebinding of the antibodies to specific cell surface markers. The cellmixture is then washed by one or more centrifugation and resuspensionsteps. The mixture is run through a FACS which separates the cells basedon different fluorescence characteristics. FACS systems are available invarying levels of performance and ability, including multi-coloranalysis. The facilitating cell can be identified by a characteristicprofile of forward and side scatter which is influenced by size andgranularity, as well as by positive and/or negative expression ofcertain cell surface markers.

Other separation techniques besides flow cytometry can also provide fastseparations. One such method is biotin-avidin based separation byaffinity chromatography. Typically, such a technique is performed byincubating cells with biotin-coupled antibodies to specific markers,such as, for example, the transmembrane protein encoded by the 103 genedescribed herein, followed by passage through an avidin column.Biotin-antibody-cell complexes bind to the column via the biotin-avidininteraction, while other cells pass through the column. The specificityof the biotin-avidin system is well suited for rapid positiveseparation. Multiple passages can ensure separation of a sufficientlevel of the TH cell subpopulation of interest.

In instances whereby the goal of the separation technique is to depletethe overall number of cells belonging to a TH cell subpopulation, thecells derived from the starting source of cells which has now beeneffectively depleted of TH cell subpopulation cells can be reintroducedinto the patient. Such a depletion of the TH cell subpopulation resultsin the amelioration of TH cell subpopulation-related disordersassociated with the activity or overactivity of the TH cellsubpopulation. Reintroduction of the TH cell subpopulation-depletedcells can be accomplished by washing the cells, resuspending in, forexample, buffered saline, and intravenously administering the cells intothe patient.

If cell viability and recovery are sufficient, TH cellsubpopulation-depleted cells can be reintroduced into patientsimmediately subsequent to separation. Alternatively, TH cellsubpopulation-depleted cells can be cultured and expanded ex vivo priorto administration to a patient. Expansion can be accomplished via wellknown techniques utilizing physiological buffers or culture media in thepresence of appropriate expansion factors such as interleukins and otherwell known growth factors.

In instances whereby the goal of the separation technique is to augmentor increase the overall number of cells belonging to a TH cellsubpopulation, cells derived from the purified TH cell subpopulationcells can be reintroduced into the patient, thus resulting in theamelioration of TH cell subpopulation-related disorders associated withan under activity of the TH cell subpopulation.

The cells to be reintroduced will be cultured and expanded ex vivo priorto reintroduction. Purified TH cell subpopulation cells can be washed,suspended in, for example, buffered saline, and reintroduced into thepatient via intravenous administration.

Cells to be expanded can be cultured, using standard procedures, in thepresence of an appropriate expansion agent which induces proliferationof the purified TH cell subpopulation. Such an expansion agent can, forexample, be any appropriate cytokine, antigen, or antibody. In the caseof TH2 cells, for example, the expansion agent can be IL-4, while forTH1 cells, the expansion agent can, for example, be IL-12.

Prior to being reintroduced into a patient, the purified cells can bemodified by, for example, transformation with gene sequences encodinggene products of interest. Such gene products should represent productswhich enhance the activity of the purified TH cell subpopulation or,alternatively, represent products which repress the activity of one ormore of the other TH cell subpopulations. Cell transformation and geneexpression procedures are well known to those of skill in the art, andcan be as those described, above, in Section 5.5.

Well known targeting methods can, additionally, be utilized in instanceswherein the goal is to deplete the number of cells belonging to aspecific TH cell subpopulation. Such targeting methods can be in vivo orin vitro, and can involve the introduction of targeting agents into apopulation of cells such that the targeting agents selectively destroy aspecific subset of the cells within the population. In vivoadministration techniques which can be followed for such targetingagents are described, below, in Section 5.10.

Targeting agents generally comprise, first, a targeting moiety which, inthe current instance, causes the targeting agent to selectivelyassociate with a specific TH cell subpopulation. The targeting agentsgenerally comprise, second, a moiety capable of destroying a cell withwhich the targeting agent has become associated.

Targeting moieties can include, but are not limited to, antibodiesdirected to cell surface markers found specifically on the TH cellsubpopulation being targeted, or, alternatively, to ligands, such asgrowth factors, which bind receptor-type molecules found exclusively onthe targeted TH cell subpopulation.

In the case of TH2 cells, for example, such a targeting moiety canrepresent an antibody directed against the extracellular portion of the103 gene product described herein, or can, alternatively, represent aligand specific for this receptor-type TH2 specific molecule. In thecase of TH1 cells, for example, such a targeting moiety can represent anantibody directed against the extracellular portion of the 200 geneproduct described herein, or can, alternatively, represent a ligandspecific for this receptor-type TH1 specific molecule.

Destructive moieties include any moiety capable of inactivating ordestroying a cell to which the targeting agent has become bound. Forexample, a destructive moiety can include, but it is not limited tocytotoxins or radioactive agents. Cytotoxins include, for example,plant-, fungus-, or bacteria-derived toxins, with deglycosylated Ricin Achain toxins being generally preferred due to their potency and lengthyhalf-lives.

5.10. Pharmaceutical Preparations and Methods of Administration

The compounds, nucleic acid sequences and TH cell subpopulation celldescribed herein can be administered to a patient at therapeuticallyeffective doses to treat or ameliorate immune disorders, e.g., TH cellsubpopulation-related disorders. A therapeutically effective dose refersto that amount of a compound or TH cell subpopulation sufficient toresult in amelioration of the immune disorder symptoms of the immunedisorder symptoms, or alternatively, to that amount of a nucleic acidsequence sufficient to express a concentration of gene product whichresults in the amelioration of the TH cell subpopulation-relateddisorders or of other immune disorders.

5.10.1. Effective Dose

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

5.10.2. Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvents can be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration (i.e.,intravenous or intramuscular) by injection, via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. It is preferredthat the TH cell subpopulation cells be introduced into patients viaintravenous administration.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice which can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

5.11. Diagnostic and Monitoring Techniques

A variety of methods can be employed for the diagnosis of immunedisorders, e.g., TH cell subpopulation-related disorders, predispositionto such immune disorders, for monitoring the efficacy of anti-immunedisorder compounds during, for example, clinical trials and formonitoring patients undergoing clinical evaluation for the treatment ofsuch disorders. Further, a number of methods can be utilized for thedetection of activated immune cells, e.g., activated members of TH cellsubpopulations.

Such methods can, for example, utilize reagents such as the fingerprintgene nucleotide sequences described in Sections 5.1, and antibodiesdirected against differentially expressed and pathway gene peptides, asdescribed, above, in Sections 5.5 (peptides) and 5.6 (antibodies).Specifically, such reagents can be used, for example, for: 1) thedetection of the presence of target gene expression, target genemutations, the detection of either over- or under-expression of targetgene mRNA relative to the non-immune disorder state or relative to anunactivated TH cell subpopulation; 2) the detection of either an over-or an underabundance of target gene product relative to the non-immunedisorder state or relative to the unactivated TH cell subpopulationstate; and 3) the identification of specific TH cell subpopulation cells(e.g., TH cells involved in an immune disorder, or activated TH cells)within a mixed population of cells.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specificfingerprint gene nucleic acid or anti-fingerprint gene antibody reagentdescribed herein, which can be conveniently used, e.g., in clinicalsettings, to diagnose patients exhibiting TH1- or TH2-relatedabnormalities.

Any cell type or tissue, preferably TH cells, in which the fingerprintgene is expressed can be utilized in the diagnostics described below.

Among the methods which can be utilized herein are methods formonitoring the efficacy of compounds in clinical trials for thetreatment of immune disorders. Such compounds can, for example, becompounds such as those described, above, in Section 5.9. Such a methodcomprises detecting, in a patient sample, a gene transcript or geneproduct which is differentially expressed in a TH cell subpopulation inan immune disorder state relative to its expression in the TH cellsubpopulation when the cell subpopulation is in a normal, or non-immunedisorder, state.

Any of the nucleic acid detection techniques described, below, inSection 5.11.1 or any of the peptide detection techniques described,below, in Section 5.11.2 can be used to detect the gene transcript orgene product which is differentially expressed in the immune disorder THcell subpopulation relative to its expression in the normal, ornon-immune disorder, state.

During clinical trials, for example, the expression of a singlefingerprint gene, or alternatively, the fingerprint pattern of a TH cellsubpopulation, can be determined for the TH cell subpopulation in thepresence or absence of the compound being tested. The efficacy of thecompound can be followed by comparing the expression data obtained tothe corresponding known expression patterns for the TH cellsubpopulation in a normal, non-immune disorder state. Compoundsexhibiting efficacy are those which alter the single fingerprint geneexpression and/or the fingerprint pattern of the immune disorder TH cellsubpopulation to more closely resemble that of the normal, non-immunedisorder TH cell subpopulation.

The detection of the product or products of genes differentiallyexpressed in a TH cell subpopulation in an immune disorder staterelative to their expression in the TH cell subpopulation when the cellsubpopulation is in a normal, or non-immune disorder, state can also beused for monitoring the efficacy of potential anti-immune disordercompounds during clinical trials. During clinical trials, for example,the level and/or activity of the products of one or more suchdifferentially expressed genes can be determined for the TH cellsubpopulation in the presence or absence of the compound being tested.The efficacy of the compound can be followed by comparing the proteinlevel and/or activity data obtained to the corresponding knownlevels/activities for the TH cell subpopulation in a normal, non-immunedisorder state. Compounds exhibiting efficacy are those which alter thepattern of the immune disorder TH cell subpopulation to more closelyresemble that of the normal, non-immune disorder TH cell subpopulation.

Given the TH2-specific nature of the 103 gene, the detection of 103 genetranscripts and/or products can be particularly suitable for monitoringthe efficacy of compounds in clinical trials for the treatment of TH2cell subpopulation-related immune disorders such as, for example, asthmaor allergy.

The expression patterns of the 105, 106 and 200 genes in TH1 cellsubpopulations relative to TH2 cell subpopulations can make thedetection of transcripts and/or products of these genes particularlysuitable for monitoring the efficacy of compounds in clinical trials forthe treatment of TH1 cell subpopulation-related immune disorders suchas, for example, multiple sclerosis, psoriasis or insulin dependentdiabetes.

Among the additional methods which can be utilized herein are methodsfor detecting TH cell responsiveness, for example, responsiveness toantigen, and for detecting activated immune cells, e.g., activatedmembers of TH cell subpopulations. Detection methods such as these areimportant in that many immune disorders involve inappropriate ratherthan insufficient immune responses. Such detection methods can be used,for example, to detect a predisposition to an immune disorder.

Methods for detecting TH cell responsiveness and/or activation cancomprise, for example, detecting in a TH cell sample a gene transcriptor product which is differentially expressed in TH cell subpopulationwhich is in an activated or responsive state (e.g., a state in which theTH cell subpopulation has been exposed to antigen), relative to a THcell subpopulation which is in an unactivated or nonresponsive state.

Any of the nucleic acid detection techniques described, below, inSection 5.11.1 or any of the peptide detection techniques described,below, in Section 5.11.2 can be used to detect such a differentiallyexpressed gene transcript or gene product.

The TH2-specific nature of the 103 gene can make the detection of itsgene transcripts and/or products particularly suitable for detectingactivation and/or responsiveness of TH2 cells. Further, the TH1-specificnature of the 105, 106 and 200 genes can make the detection oftranscripts and/or products of these genes particularly suitable for thedetection of TH1 activation and/or responsiveness.

5.11.1 Detection of Fingerprint Gene Nucleic Acids

DNA or RNA from the cell type or tissue to be analyzed can easily beisolated using procedures which are well known to those in the art.Diagnostic procedures can also be performed "in situ" directly upon, forexample tissue sections (fixed and/or frozen) of patient tissue obtainedfrom biopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described in Section 5.4can be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocolsand Applications", Raven Press, NY). Expression of specific cells withina population of cells can also be determined, via, for example, in situtechniques such as those described above, or by standard flow cytometrictechniques.

Fingerprint gene nucleotide sequences, either RNA or DNA, can, forexample, be used in hybridization or amplification assays of biologicalsamples to detect TH cell subpopulation-related disorder gene structuresand expression. Such assays can include, but are not limited to,Southern or Northern analyses, single stranded conformationalpolymorphism analyses, in situ hybridization assays, and polymerasechain reaction analyses. Such analyses can reveal both quantitativeaspects of the expression pattern of the fingerprint gene, andqualitative aspects of the fingerprint gene expression and/or genecomposition. That is, such techniques can detect not only the presenceof gene expression, but can also detect the amount of expression,particularly which specific cells are expressing the gene of interest,and can, further, for example, detect point mutations, insertions,deletions, chromosomal rearrangements, and/or activation or inactivationof gene expression.

Diagnostic methods for the detection of fingerprint gene-specificnucleic acid molecules can involve for example, contacting andincubating nucleic acids, derived from the cell type or tissue beinganalyzed, with one or more labeled nucleic acid reagents as aredescribed in Section 5.4, under conditions favorable for the specificannealing of these reagents to their complementary sequences within thenucleic acid molecule of interest. Preferably, the lengths of thesenucleic acid reagents are at least 15 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid:fingerprint molecule hybrid. The presence of nucleic acids from thecell type or tissue which have hybridized, if any such molecules exist,is then detected. Using such a detection scheme, the nucleic acid fromthe tissue or cell type of interest can be immobilized, for example, toa solid support such as a membrane, or a plastic surface such as that ona microtiter plate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described inSection 5.4 are easily removed. Detection of the remaining, annealed,labeled fingerprint nucleic acid reagents is accomplished using standardtechniques well-known to those in the art.

Alternative diagnostic methods for the detection of fingerprint genespecific nucleic acid molecules can involve their amplification, e.g.,by PCR (the experimental embodiment set forth in Mullis, K. B., 1987,U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh, D. Y et al., 1989, Proc.Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal., 1988, Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA can be isolated include any tissue in which wild typefingerprint gene is known to be expressed, including, but not limited,to TH0, TH1 and/or TH2 cell type-containing tissues. A sequence withinthe cDNA is then used as the template for a nucleic acid amplificationreaction, such as a PCR amplification reaction, or the like. The nucleicacid reagents used as synthesis initiation reagents (e.g., primers) inthe reverse transcription and nucleic acid amplification steps of thismethod are chosen from among the fingerprint gene nucleic acid reagentsdescribed in Section 5.4. The preferred lengths of such nucleic acidreagents are at least 9-30 nucleotides. For detection of the amplifiedproduct, the nucleic acid amplification can be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product can be made such that the product can bevisualized by standard ethidium bromide staining or by utilizing anyother suitable nucleic acid staining method.

In addition to methods which focus primarily on the detection of onefingerprint nucleic acid sequence, fingerprint patterns can also beassessed in such detection schemes. Fingerprint patterns, in thiscontext, contain the pattern of mRNA expression of a series (i.e., atleast two and up to the total number present) of fingerprint genesobtained for a given tissue or cell type under a given set ofconditions. Such conditions can include, for example, TH cellsubpopulation-related disorders, and conditions relevant to processesinvolved in the differentiation, maintenance and effector function of THcell subpopulations.

TH1-related disorders can include, for example, chronic inflammatorydiseases and disorders, such as Crohn's disease, reactive arthritis,including Lyme disease, insulin-dependent diabetes, organ-specificautoimmunity, including multiple sclerosis, Hashimoto's thyroiditis andGrave's disease, contact dermatitis, psoriasis, graft rejection, graftversus host disease and sarcoidosis. TH2-related disorders can include,for example, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis) and certainviral infections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

Fingerprint patterns can be generated, for example, by utilizing adifferential display procedure, as discussed, above, in Section 5.1.1.2,Northern analysis and/or RT-PCR. Any of the gene sequences described,above, in Section 3.2.1 can be used as probes and/or RT-PCR primers forthe generation and corroboration of such fingerprint patterns.

5.11.2 Detection of Target Gene Peptides

Antibodies directed against wild type or mutant fingerprint genepeptides, which are discussed, above, in Section 5.6, can also be usedas TH cell subpopulation-related disorder diagnostics and prognostics,as described, for example, herein. Such diagnostic methods, can be usedto detect fingerprint gene product, abnormalities in the level offingerprint gene protein expression, or abnormalities in the structureand/or temporal, tissue, cellular, or subcellular location offingerprint gene protein. Structural differences can include, forexample, differences in the size, electronegativity, or antigenicity ofthe mutant fingerprint gene protein relative to the normal fingerprintgene protein.

Protein from the tissue or cell type to be analyzed can easily beisolated using techniques which are well known to those of skill in theart. The protein isolation methods employed herein can, for example, besuch as those described in Harlow and Lane (Harlow, E. and Lane, D.,1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), which is incorporated herein byreference in its entirety.

Preferred diagnostic methods for the detection of wild type or mutantfingerprint gene peptide molecules can involve, for example,immunoassays wherein fingerprint gene peptides are detected by theirinteraction with an anti-fingerprint gene product-specific antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.6, useful in the present invention can beused to quantitatively or qualitatively detect the presence of wild typeor mutant fingerprint gene peptides. This can be accomplished, forexample, by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below, this Section,) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection. Such techniquesare especially preferred if the fingerprint gene peptides are expressedon the cell surface, such as, for example, is the case with the 10 geneproduct, the 200 gene product and the transmembrane form of 103 geneproduct. Thus, the techniques described herein can be used to detectspecific cells, within a population of cells, which express thefingerprint gene product of interest.

The antibodies (or fragments thereof) useful in the present inventioncan, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of fingerprint genepeptides. In situ detection can be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for wild type or mutant fingerprint gene peptides typicallycomprise incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells, or cells which have beenincubated in tissue culture, in the presence of a detectably labeledantibody capable of identifying fingerprint gene peptides, and detectingthe bound antibody by any of a number of techniques well-known in theart.

The biological sample can be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support can then be washed with suitable buffersfollowed by treatment with the detectably labeled fingerprintgene-specific antibody. The solid phase support can then be washed withthe buffer a second time to remove unbound antibody. The amount of boundlabel on solid support can then be detected by conventional means.

By "solid phase support or carrier" is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or mutantfingerprint gene product antibody can be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

One of the ways in which the fingerprint gene peptide-specific antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., "The Enzyme LinkedImmunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E.,1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, ENZYMEIMMUNOASSAY, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al., (eds.),1981, ENZYME IMMUNOASSAY, Kgaku Shoin, Tokyo). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵² Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

6. Example: Identification and Characterization of a TH2-Enriched Gene

In the Example presented in this Section, the transgenic T cell paradigmdescribed, above, in Section 5.1.1.1, was utilized to identify a gene,designated herein as the 102 gene, which is expressed in TH2 cells. Theidentified gene is present in TH2 cells at a much higher level than inTH1 cells. Thus, the Example presented herein demonstrates theusefulness of the paradigm approach of the invention for theidentification of genes that are differentially expressed in TH cellsubpopulations.

6.1 Materials and Methods

Transgenic mice: Naive CD4⁺ cells were obtained from the spleens and/orlymph nodes of unprimed transgenic mouse strains harboring a T cellreceptor (TCR) recognizing ovalbumin (Murphy et al., 1990, Science250:1720).

Ova-specific transgenic T cells: Suspensions of ova-specific T cellswere co-cultured with stimulatory peptide antigen and antigen presentingcells essentially as described in Murphy et al. (Murphy et al., 1990,Science 250:1720). Briefly, 2-4×10⁶ T cells were incubated withapproximately twice as many TA3 antigen presenting cells in the presenceof 0.3 μM Ova peptide. TH1 cultures contained approximately 10 ng/mlrecombinant mIL-12. Conversely, TH2 cells received IL-4 (1000 u/ml).Cultures were harvested at various time points after initiation ofculture. T cells were purified of TA3 cells using anti-CD4 coatedmagnetic beads (Dynal, Inc.). T cells were pelleted by gentlecentrifugation and lysed in the appropriate volume of RNAzol™ (Tel-Test,Friendswood, Tex.).

Tissue collection and RNA isolation: Cells were quick frozen on dry ice.Samples were then homogenized together with a mortar and pestle underliquid nitrogen.

Total cellular RNA was extracted from tissue with either RNAzol™ orRNAzolB™ (Tel-Test, Friendswood, Tex.), according to the manufacturer'sinstructions. Briefly, the tissue was solubilized in an appropriateamount of RNAzol™ or RNAzolB™, and RNA was extracted by the addition of1/10 v/v chloroform to the solubilized sample followed by vigorousshaking for approximately 15 seconds. The mixture was then centrifugedfor 15 minutes at 12,000 g and the aqueous phase was removed to a freshtube. RNA was precipitated with isopropanol. The resultant RNA pelletwas dissolved in water and re-extracted with an equal volume ofchloroform to remove any remaining phenol. The extracted volume wasprecipitated with 2 volumes of ethanol in the presence of 150 mM sodiumacetate. The precipitated RNA was dissolved in water and theconcentration determined spectroscopically (A₂₆₀).

Differential display: Total cellular RNA (10-50 μg) was treated with 20Units DNase I (Boehringer Mannheim, Germany) in the presence of 40 Unitsribonuclease inhibitor (Boehringer Mannheim, Germany). After extractionwith phenol/chloroform and ethanol precipitation, the RNA was dissolvedin DEPC (diethyl pyrocarbonate)-treated water.

Differential mRNA display was carried out as described, above, inSection 5.1.1.2. RNA (0.4-2 μg) was reverse-transcribed usingSuperscript reverse transcriptase (GIBCO/BRL). The cDNAs were thenamplified by PCR on a Perkin-Elmer 9600 thermal cycler. The reactionmixtures (20 μl) included arbitrary decanucleotides and one of twelvepossible T₁₁ VN sequences, wherein V represents either dG, dC, or dA,and N represents either dG, dT, dA, or dC. Parameters for the 40 cyclePCR were as follows: Hold 94° C. 2 minutes; Cycle 94° C. 15 seconds, 40°C. 2 minutes; Ramp to 72° 30 seconds; Hold 72° C. 5 minutes; Hold 4° C.

Radiolabelled PCR amplification products were analyzed byelectrophoresis on 6% denaturing polyacrylamide gels.

Reamplification and subcloning: PCR bands of interest were recoveredfrom sequencing gels and reamplified.

Briefly, autoradiograms were aligned with the dried gel, and the regioncontaining the bands of interest was excised with a scalpel. The excisedgel fragment was eluted by soaking in 100 μl TE (Tris-EDTA) buffer atapproximately 100° C. for 15 minutes. The gel slice was then pelleted bybrief centrifugation and the supernatant was transferred to a newmicrocentrifuge tube. DNA was combined with ethanol in the presence of100 mM Sodium acetate and 30 μg glycogen (Boerhinger Mannhein, Germany)and precipitated on dry ice for approximately 10 minutes. Samples werecentrifuged for 10 minutes and pellets were washed with 80% ethanol.Pellets were resuspended in 10 μl distilled water.

5 μl of the eluted DNA were reamplified in a 100 μl reaction containing:standard Cetus Taq polymerase buffer, 20 μM dNTPs, 1 μM of each of theoligonucleotide primers used in the initial generation of the amplifiedDNA. Cycling conditions used were the same as the initial conditionsused to generate the amplified band, as described above. One-half of theamplification reaction was run on a 2% agarose gel and eluted usingDE-81 paper (Whatman Paper, Ltd., England) as described in Sambrook etal., supra. Recovered fragments were ligated into the cloning vectorpCR™II (Invitrogen, Inc., San Diego Calif.) and transformed intocompetent E. coli strain DH5α (Gibco/BRL, Gaithersburg, Md.). Colonieswere grown on LB-agar plates containing ampicillin (100 μg/ml) and X-gal(40 μg/ml) to permit blue/white selection.

Sequence analysis: After subcloning, reamplified cDNA fragments weresequenced on an Applied Biosystems Automated Sequencer (AppliedBiosystems, Inc. Seattle, Wash.). Sequence was obtained from four ormore independent transformants containing the same insert. Thenucleotide sequence shown herein represents either the consensus of theinformation obtained from the four sequences, or the sequence obtainedfrom a representative clone, as indicated. Such primary sequence datawas edited and trimmed of vector sequences and highly repetitivesequences and used to search Genbank databases using the BLAST(Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410) program.

Northern analysis: RNA samples were electrophoresed in a denaturingagarose gel containing 1-1.5% agarose (SeaKem™ LE, FMC BioProducts,Rockland, Me.) containing 6.3% formaldehyde. Samples containing 5-20 μgof total RNA were mixed with denaturing loading solution (72% deionizedformamide and bromophenol blue) and heated to 70° C. for 5 minutes.Samples were placed on ice and immediately loaded onto gels. Gels wererun in 1×MOPS buffer (100 mM MOPS, 25 mM sodium acetate, 5 mM EDTA).After electrophoresis, the gels were stained with ethidium bromide andvisualized with ultraviolet light.

After completion of electrophoresis, gels were soaked in 50 mM sodiumhydroxide with gentle agitation for approximately 30 minutes to lightlycleave RNA. Gels were rinsed twice in water and then neutralized bysoaking in 0.1 M Tris-HCl (pH 7.5) for approximately 30 minutes. Gelswere briefly equilibrated with 20×SSC (3 M sodium chloride, 0.3 M sodiumcitrate) and then transferred to nylon membranes such as Hybond™,-N,(Amersham, Inc., Arlington Heights, Ill.) or Zeta-Probe (Bio-Rad, Inc.,Hercules, Calif.) overnight in 20×SSC. Membranes containing transferredRNA were baked at 80° C. for 2 hours to immobilize the RNA.

DNA fragments to be used as probes were of various sizes and werelabeled using a random hexamer labeling technique. Briefly, 25 ng of apurified DNA fragment was used to generate each probe. Fragments wereadded to a 20 μl random hexanucleotide labeling reaction (BoehringerMannhein, Inc., Indianapolis, Ind.) containing random hexamers and a mixof the nucleotides dCTP, dGTP, and dTTP (at a final concentration of 25μM each). The reaction mix was heat-denatured at 100° C. for 10 minutesand then chilled on ice. 5 μl of α-³² P-dATP (50 μCi; Amersham, Inc.,Arlington Heights, Ill.) and Klenow DNA polymerase (2 units; BoehringerMannheim, Inc., Indianapolis, Ind.) were added. Reactions were incubatedat 37° for 30 minutes. Following incubation, 30 μl water was added tothe labeling reaction and unincorporated nucleotides were removed bypassing the reactions through a BioSpin-6™ chromatography column(Bio-Rad, Inc., Hercules, Calif.). Specific incorporation was determinedusing a scintillation counter. 1-5×10⁶ cpm were used per mlhybridization mixture.

Nylon membranes containing immobilized RNA were prehybridized accordingto manufacturer's instructions. Radiolabelled probes were heat denaturedat 70° C. in 50% deionized formamide for 10 minutes and ten added to thehybridization mixture (containing 50% formamide, 10% dextran sulfate,0.1% SDS, 100 μg/ml sheared salmon sperm DNA, 5×SSC, 5×Denhardt'ssolution, 30 mM Tris-HCl (pH 8.5), 50 mM NaPO₄ (pH 6.5). Hybridizationswere carried out at 42° C. overnight. Nylon membranes were then bathedfor 2 minutes in a wash solution of 0.2×SSC and 0.1% SDS at roomtemperature to remove most of the remaining hybridization solution. Themembranes were then bathed twice in fresh 42° C. preheated wash solutionfor 20 minutes. Filters were covered in plastic wrap and exposed toautoradiographic film to visualize results.

6.2 Results

A transgenic T cell paradigm (as described, above, in Section 6.1) wasutilized to identify genes which are differentially expressed betweenTH1 and TH2 cells.

RNA samples were isolated from TH1 and TH2 cell populations after eithersecondary or tertiary antigen stimulation. The samples were thenanalyzed via differential display techniques. FIG. 1 shows amplifiedfragments obtained from these samples, with the arrow indicating a PCRproduct, designated band 102, which was judged to represent a cDNAderived from RNA produced by a gene which is expressed at a higher levelin TH2 cell subpopulations, relative to TH1 cell subpopulations. Thegene corresponding to band 102 is referred to herein as the 102 gene.

The amplified band 102 cDNA was recovered, reamplified, subcloned into acloning vector and sequenced, as described, above, in Section 6.1. Thenucleotide sequence (SEQ ID NO:1) of a representative band 102 clone,specifically, clone 102.1, is shown in FIG. 2.

A BLAST (Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410)database search with this consensus sequence resulted in an alignmentwith 98% identity to the mouse Granzyme A, or Hanukah factor, gene,(Masson, D. et al., 1986, FEBS Lett. 208:84-88; Masson, D. et al., 1986,EMBO J. 5:1595-1600; Gershenfeld, H. K. and Weissman, I. L., 1986,Science 232:854-858), which encodes a trypsin-like serine protease. Thehuman homolog of this gene is also known (Gershenfeld, H. K. et al.,1988, Proc. Natl. Acad. Sci. USA 85:1184-1188).

To confirm the gene's putative differential regulation, amplified band102 cDNA was used to probe Northern RNA blots containing RNA samplesfrom TH1 and TH2 cell lines, and from spleen and thymus tissue. FIG. 3shows the results of one such Northern blot analysis, in which thesteady state level of message for 102 gene mRNA are significantlyincreased in RNA samples derived from stimulated TH2 versus TH1 samples.Further, the positive signals in both thymus and spleen RNA samplessupports the indication that the 102 gene product is involved in someaspect of T cell function. Thus, the Northern analysis confirmed theputative differential TH2 regulation which had been suggested by thedifferential display result.

Therefore, by utilizing the transgenic T cell paradigm described in thisSection and in Section 5.1.1.1, above, a TH2 differentially regulatedgene, designated here the 102 gene, and corresponding to the mouseGranzyme A/Hanukah factor gene, has been identified, therebycorroborating the usefulness of such paradigms in identifying genesexpressed preferentially in T helper cell subpopulations such as TH1 orTH2 cell populations.

Further, while the gene identified here had previously been found to beexpressed in natural killer T cells and, further, had been reported tobe expressed in a fraction of CD4⁺ cells (Fruth, U. et a;., 1988, Eur.J. Imm. 18:773-781; Liu, C. C. et al., 1989, J. Exp. Med.170:2105-2118), the results described herein represent the firstinstance in which a TH cell subpopulation role for this gene has beenfound. Prior to this study, the gene had been reported to be expressedin T cells in a variety of situations, including TH1 cell subpopulation-and TH2 cell subpopulation-related disorders. For example, GranzymeA/Hanukah factor expression has been reported in allograft rejection(Muller, C. et al., 1988, J. Exp. Med. 167:1124-1136) and autoimmunediseases (Ojcius, D. M. and Young, D. E., 1990, Cancer Cells 2:138-145;Young, L. H. Y. et al., 1992, Am. J. Path. 140:1261-1268), which are TH1cell subpopulation-related disorders, and also in Leishmania infectionsusceptible mice (Moll, H. et al., 1991, Inf. and Imm. 59:4701-4705) andin leprosy lesions (Ebnet, K. et al., 1991, Int. Imm. 3:9-19; Cooper, C.L. et al., 1989, J. Exp. Med. 169:1565-1581), which are both TH2 cellsubpopulation-related disorders.

The differential TH2-like expression demonstrated here represents,therefore, the first molecular evidence clearly indicating a moreprimary role for the gene product in TH2 versus TH1 cell subpopulations.

7. Example: Identification and Characterization of a TH2-Specific Gene

In the Example presented in this Section, the transgenic T cellparadigm, described, above, in Sections 5.1.1.1 and 6, was utilized toidentify a gene which is differentially expressed in TH2 cells.Specifically, this gene is present in TH2 cells while being completelyabsent from TH1 cells. The gene, which corresponds to a gene known,alternatively, as ST-2, T1 and Fit-1, does not appear to be expressed inany other assayed cell type or tissue, and is demonstrated here for thefirst time to encode a marker which is, in vivo, completelyTH2-specific. The 103 gene encodes a cell surface protein, the potentialsignificance of which is discussed herein.

7.1 Materials and Methods

RT-PCR analysis: Quantitative RT-PCR was performed as follows. 1-2 μg oftotal RNA, prepared as described, above, in Section 6.1, was reversetranscribed with oligo dT.sub.(12-18) primers and Superscript™ RNAase H⁻reverse transcriptase (Gibco-BRL, Gaithersburg, Md.). Briefly, RNA wascombined with 1 μl oligo dT (500 μg/ml) in a total volume of 11 μl. Themixture was heated to 70° C. for 10 minutes and chilled on ice. After abrief centrifugation, RNA was reverse transcribed for 1 hour. Aliquotsof the first strand cDNA were stored at -20° C. until just prior to use.

Expression levels were determined by PCR amplification of serialdilutions of first strand cDNA. In this procedure, cDNA is seriallydiluted in water. The dilutions are then batch amplified by PCR usingsequence-specific primers. All PCR reactions are amplified underidentical conditions. Therefore, the amount of product generated shouldreflect the amount of sequence template which was initially present.5-10 fold dilutions of cDNA were used and enough dilutions were usedsuch that the amount of product subsequently produced ranged fromclearly visible, by UV illumination of ethidium bromide-stained gels, tobelow detection levels. The method described herein can distinguish10-fold differences in expression levels.

Primers were designed for the amplification of the sequenced amplifiedbands, which were chosen using the program OLIGO (National Biosciences,Plymouth, Minn.). Primer sequences used in this assay were as follows:and 103 sense primer, 5'-TTGCCATAGAGAGACCTC-3' (SEQ ID NO:18); band 103antisense primer, 5'-TGCTGTCCAATTATACAGG-3' (SEQ ID NO:19); murine gammaactin sense primer, 5'-GAACACGGCATTGTCACTAACT-3' (SEQ ID NO:20); murinegamma actin antisense primer, 5'-CCTCATAGATGGGCACTGTGT-3' (SEQ IDNO:21).

All quantitative PCR reactions were carried out in a 9600 Perkin-ElmerPCR machine (Perkin-Elmer). Generally, amplification conditions were asfollows: 30-40 cycles consisting of a 95° C. denaturation for 30seconds, 50-60° C. annealing for 30 seconds, and 72° C. extension for 1minute. Following cycling, reactions were extended for 10 minutes at 72°C.

RNase Protection Assays: RNAse protection assays were performedaccording to manufacturer's instructions, using a kit purchased fromAmbion, Inc. RNA probes derived from GenBank Accession No. Y07519 wereutilized in the RNAse protection assays. These probes were alsogenerated according to manufacturer's instructions, using a kitpurchased from Ambion, Inc. The sequence of these RNA probes correspondsto the 5' end of the gene, and includes both coding and 5' untranslatedsequences.

Anti CD-3 stimulation: Conditions were as described, below, in Section8.1.

Other procedures: All other cell sample collection, RNA isolation,differential display, sequence analysis, and Northern proceduresperformed in the experiments described in this Example were asdescribed, above, in Section 6.1.

7.2 Results

A differential display analysis of RNA isolated from TH1 and TH2 cellsamples obtained from a transgenic T cell paradigm study as described,above, in Section 6.1. Specifically, TH cells were obtained fromtransgenic mice harboring a T cell receptor recognizing ovalbumin(Murphy et al., 1990, Science 250:1720) were stimulated three times, andRNA was obtained from TH1 and TH2 cells. Differential display analysisof the RNA samples resulted in the identification of a TH2differentially expressed band, designated and referred to herein as band103. The gene corresponding to band 103 is referred to herein as the 103gene.

103 gene cDNA was isolated, amplified and subcloned, and nucleotidesequence (SEQ ID NO:2) was obtained, as shown in FIG. 4A. A databasesearch revealed that the nucleotide sequence of band 103 resulted in analignment with 98% identity to the mouse form of a gene known,alternatively, as the ST-2, T1 or Fit-1 gene (Klemenz, R. et al., 1989,Proc. Natl. Acad. Sci. USA 86:5708-5712; Tominaga, S., 1989, FEBS Lett.258:301-301; Werenskiold, A. K. et al., 1989, Mol. Cell.Biol.9:5207-5214; Werenskiold, A. K., 1992, Eur. J. Biochem.204:1041-1047; Yanagisawa, K. et al., 1993, FEBS Lett. 318:83-87;Bergers, G. et al., 1994, EMBO J. 13:1176-1188).

The 103 gene encodes, possibly via alternatively spliced transcripts,transmembrane and soluble forms of proteins which belong to theimmunoglobulin superfamily. The soluble form of the protein shows a highlevel of similarity to the extracellular portion of the mouseinterleukin-1 receptor type 1 (IL-1R1) and interleukin-1 receptor type 2(IL-1R2; which lacks a cytoplasmic domain), while the transmembraneportion (termed ST2L) bears a high resemblance to the entire IL-1R1sequence and to the extracellular IL-1R2 sequences. Further, the 103gene appears to be tightly linked to the interleukin 1 receptor-type 1locus (McMahn, C. J. et al., 1991, EMBO J. 10:2821-2832; Tominaga, S. etal., 1991, Biochem. Biophys. Acta. 1090:1-8). Additionally, the human103 gene homolog has also been reported (Tominaga, S. et al., 1992,Biochem. Biophys. Acta. 1171:215-218). FIG. 4B illustrates the 103 genetransmembrane and soluble forms of protein, and shows their relationshipto the IL-1R1 protein sequence.

A quantitative RT-PCR analysis (FIG. 5) of RNA obtained from cells of aTH1 and TH2 cells, generated as described above, 24 hours after tertiaryantigen stimulation not only confirmed the putative TH2 differentialexpression of the gene, but, revealed that the expression of the 103gene appears to be TH2 specific, i.e., the sensitive RT-PCR studydetected no 103 gene message in the TH1 RNA sample.

The TH2 specificity of the 103 gene was further confirmed by a Northernanalysis of several representative TH cell lines. Specifically, threeTH2 clones (CDC25, D10.G4, DAX) and three TH1 clones (AE7.A, Dorris,D1.1) were utilized and RNA samples were isolated from eitherunstimulated cells or from cells which had been stimulated for 6 hourswith plate-bound anti-CD3 antibody. The samples were probed with band103 sequences, as shown in FIG. 6. While 103 gene RNA is present in RNAobtained from both unstimulated and stimulated cells of each of the TH2cell lines, 103 gene RNA is completely absent from all of the samplesobtained from either stimulated or unstimulated TH1 cells. As the RT-PCRanalysis described above first demonstrated, the 103 gene appears to beTH2 specific, with no detectable TH1-derived signal being present.

The data presented in FIG. 7 represent an additional Northern analysisin which 103 gene expression was assayed in TH cell clones (lanes 1-5)and in murine tissues (lanes 6-10). In addition to corroborating theexpression of 103 gene RNA in both stimulated and unstimulated TH2cells, the data presented here demonstrate that 103 gene expressionappears to be negative in each of the tissues (i.e., brain, heart, lung,spleen, and liver) tested.

FIG. 8 illustrates an RNAse protection assay which demonstrates twopoints regarding 103 gene regulation. First, this analysis of TH cellclones confirms the TH2-specific results described, above. Specifically,the results of this study demonstrate by RNase protection, that 103 genemRNA is absent from the TH1 clone AE7, but is present in the TH2 cloneD10.G4.

Second, RNAse protection revealed that alternate forms of 103 genetranscripts are produced upon stimulation of TH2 clones. Specifically,within 6 hours of anti-CD3 stimulation, two additional forms of 103 genetranscript appear in TH2 clones. These additional 103 gene transcriptforms represent, one, a transcript encoding a shortened, secreted,soluble form of the band 103 gene product, and, two, a smaller, termedmini, transcript which encodes a yet shorter form of the gene product.Thus, it appears that, while the 103 gene transcript encoding thetransmembrane gene product is expressed in both unstimulated andstimulated TH2 cells, the two shorter forms of transcript are expressedin a TH2-specific inducible manner. Further, while the 103 genetranscript encoding the transmembrane product are expressed in bothstimulated and unstimulated TH2 cells, the level of this transcriptpresent in stimulated is lower, i.e., is downregulated. Thus, the lowerlevel of transmembrane product and higher level of secreted 103 geneproduct can act synergistically to dampen some stimulation-inducedsignal transduction event.

Additionally, it should be noted that the results presented hereinrepresent the first time the mini form of 103 gene transcript, which canencode a shorter version of the soluble form of 103 gene product, hasbeen observed.

To summarize, while 103 gene expression in T helper cell lines hadpreviously been reported (Tominaga, S. et al., 1992, Biochem. Biophys.Acta. 1171:215-218), the TH paradigm/differential display techniquesutilized here have demonstrated, for the first time, that the 103 geneencodes a TH2 cell subpopulation-specific surface marker. In fact, theresults described in this Example demonstrate that the firstidentification of any in vivo TH cell subpopulation-specific cellularmarker.

Given its status as both a TH2 cell subpopulation-specific marker andcell surface protein, the full length 103 gene product can be utilizedin a variety of methods to modulate TH cell subpopulation-relateddisorders and/or to identify compounds which exhibit such modulatorycapability. The truncated forms of the 103 gene products can,additionally, be used as part of these methods. Modulatory methods aredescribed, above, in Section 5.9, while strategies for theidentification of modulatory compounds are described, above, in Section5.8.

8. Example: Identification of Novel TH Cell Subpopulation DifferentiallyExpressed Genes

In the Example presented in this Section, novel gene sequencesrepresenting genes which are differentially expressed in TH cellsubpopulations and/or during the differentiation of such subpopulationsare described.

8.1 Materials and Methods

T cell clone paradigm: T cell clone paradigm searches were conducted asdescribed, above, in Section 5.1.1.1. Specifically, the TH cell cloneparadigms used three different clones: D10.G4 (TH2), AE7 (TH1) and D1.1(TH1). Prior to stimulation, cell cultures were enriched for live cellsby centrifugation through a Ficoll gradient. Recovered cells werecounted and their viability was examined using trypan blue exclusion.Cells were replated into either T25 or T75 flasks at approximately 5×10⁶cells in 5 mls or 1.5×10⁶ cells in 10 mls of culture medium,respectively.

Coating was performed, generally, according to Current Protocols inImmunology, 1992, Coligan, J. E. et al., John Wiley & Sons, NY, pp3.12.4-3.12.6). Specifically, prior to plating, the flasks were coatedwith anti-CD3-ε antibodies (hybridoma supernatant from the 145-C11hybridoma; Parmingen, Inc., San Diego Calif.). For coating, antibodieswere resuspended in PBS at 1-2 μg/ml at a volume sufficient to coat thebottom of the flasks. Coating solution was incubated on the flasks forat least one hour at 37° C.

After incubation, the antibody coating solution was removed byaspiration and cells were immediately added. Flasks were placed in a 37°C. incubator for 6 hours. Cells were harvested by, for example, removalof supernatant from the culture, followed by direct lysing of cells byaddition of RNAzol™ solution. cDNA was produced as described below.

cDNA isolation: RNA was harvested from cells using techniques described,above, in Section 6.1. mRNA was purified directly, using a QuickPrep™mRNA Purification Kit (Pharmacia) according to manufacturer'sinstructions.

The TH1 cDNA library was constructed using a Gibco BRL SuperScript™Lambda System Kit, according to manufacturer's instructions. Briefly,4.5 μg of purified mRNA was used as starting material for the synthesisof poly A-primed first strand cDNA containing a Not-1 cloning site.Second strand cDNA synthesis was performed with RNAse H treatmentfollowed by random priming. Sal-1 adaptors were ligated to the 5' end ofthe resulting double-stranded cDNA. The ligated cDNA was digested withNot-1 and size fractionated. Fractions containing cDNAs within the sizerange of 0.5 to 8.0 kb in length were cloned into Sal-1/Not-1 λZipLox™arms. Recombinant phage was then packaged using the Stratagene Gigapack™II Packaging Extracts Kit, according to manufacturer's instructions. E.coli strain Y 1090(ZL)™ (Gibco BRL) cells were transformed with packagedrecombinant phage and plated at a density of 50,000 pfu per 150 mm dish.Plaques were screened by hybridization to a radiolabelled probegenerated from a subcloned band 200 cDNA fragment. Excision of cDNAinserts from lambda clones and introduction of the recombinant plasmidDNA into E. coli DH10B(ZIP)™ (Gibco BRL) was performed according tomanufacturer's instructions.

For isolation of 200 gene cDNAs, the cDNA library was screened with aprobe generated by labeling the entire sequence of the band 200 subcloneO, which was constructed using amplified DNA obtained from thedifferential display analysis. The band 200 sequence was excised fromthe pCRII Cloning Vector™ (Invitrogen) by digestion with EcoRI.Approximately 1/100,000 cDNA library plaques were scored as positivewhen screened with this probe. Several clones, including 200-P and200AF, were chosen for further study.

The cDNA library described above was also used to isolate 54 gene cDNAclones. For screening, the entire excised band 54 insert was used as aprobe.

Other procedures: All transgenic T cell manipulations, cell samplecollection, additional RNA isolation, differential display, sequenceanalysis, and Northern procedures performed in the experiments describedin this Example were as described, above, in Section 6.1.

8.2 Results

Transgenic T cell paradigm and T cell clone paradigm searches wereconducted to identify gene sequences which represent genesdifferentially expressed within and/or among TH cell subpopulationsand/or during the differentiation of such subpopulations. Describedherein are several novel genes which have been identified via theseparadigm searches. Specifically, the genes described herein have beendesignated the 10, 54, 57, 105, 106, 161 and 200 genes. A summary of thedifferential expression characteristics of the novel gene sequencesdescribed herein is presented in Table 1, above.

The band 10 and 57 have been identified as TH inducible gene sequences.That is, the expression of such genes in unstimulated TH cells is eitherundetectable or is detectable at extremely low levels, but isupregulated in both stimulated TH1 and TH2 cells. In fact, the 10 geneexpression is detectable as early as 6 hours post stimulation. Thus,such gene products can be involved in the activation of TH cells and/orcan be involved in the maintenance of mature TH cell function, in anon-TH cell subpopulation-specific manner.

FIG. 9 depicts the nucleotide sequence (SEQ ID NO:3) of the 10 genecoding region and the derived amino acid sequence of the 10 gene product(SEQ ID NO:9). While database searches reveal that the 10 gene sequenceis novel, that is, has not previously been reported in the databases, ananalysis of the portion of the 10 gene corresponding to the band 10nucleotide sequence (the underlined portion of the nucleotide sequenceof FIG. 9) shows, as depicted in FIG. 10A-C, a high similarity to aspecific class of known gene products. Specifically, as thehydrophilicity plots of FIG. 10A-C show, the 10 gene product appears toencode a protein having a seven transmembrane domain sequence motif.Interestingly, the gene products belonging to this class of protein tendto represent G protein-coupled receptor molecules. (See, e.g.,Larhammar, D. et al., 1992, J. Biol. Chem. 267: 10935-10938; Law, S. F.et al., 1991, J. Biol. Chem. 266: 17885-17897.) Thus, the TH inducibleexpression of the 10 gene coupled with the predicted protein structureof its gene product, suggests that the 10 gene product is involved in asignal transduction event important to the differentiation of mature THcells.

Additionally, as the map shown in FIG. 11 indicates, the chromosomallocation of the murine 10 gene has been identified. The 10 gene locus islocated on Chromosome 12, is closely linked to a class of genes encodingT cell autoantigens, and additionally, maps near the Ig heavy chain genelocus.

The nucleotide sequence (SEQ ID NO:4) of a representative band 57 cloneis depicted in FIG. 12. The gene corresponding to band 57 is the 57gene. The 57 gene appears to be a novel gene sequence in that it doesnot appear within the published databases. No homology to known peptidedomains has, thus far, been identified.

As shown in Table 1, above, the genes 105, 106 and 200 are eachexpressed at a higher level within the TH1 cell subpopulation, asrevealed by the TH1 differential appearance of amplified bands 105, 106and 200. Nucleotide sequences contained within bands 105 and 106 aredepicted in FIGS. 13 (SEQ ID NO:5) and 14 (SEQ ID NO:6), respectively.As discussed below, the sequence of the 200 gene is depicted in FIG. 17(SEQ ID NO:8). None of these sequences appear within publisheddatabases. Given the TH1-specific expression pattern each of thesesequences exhibits, the genes and their gene products can potentially beused as treatments for TH1-related disorders, as diagnostics for suchdisorders, and/or as part of methods for the identification of compoundscapable of ameliorating TH1-related disorders.

The 161 gene appears to be TH cell subset specific. That is, 161 geneexpression has been observed in either TH1 cells or in TH2 cells, butits expression has never been observed, simultaneously, in both TH1 andTH2 cell subpopulations. The details of the 161 gene differentialexpression pattern are currently being elucidated. It is possible that161 gene expression is indicative of the presence of yet another TH cellsubpopulation, in addition to TH1, TH2 and TH0 cell subpopulations.

FIG. 15 presents the band 161 nucleotide sequence. While the 161 geneappears to be a novel sequence, it bears a distinct level of similarityto a set of gene sequences (SEQ ID NOS:13-17) in published databases, asshown in FIG. 16. Interestingly, the genes within this group eachcontain alpha-interferon responsive promoters.

Band 200 was utilized as a probe to identify and isolate 200 gene cDNAclones, including clones designated 200-P and 200-AF, which have beendeposited with the NRRL, as summarized in Section 10, below. The cDNAclones were characterized, yielding the full length nucleotide sequence(SEQ ID NO:8) of the 200 gene coding region, as shown in FIG. 17. FIG.17 also depicts the 200 gene product derived amino acid sequence (SEQ IDNO:10). Database searches reveal that the 200 gene product is a novelreceptor which contains an extracellular Ig domain, thus placing itwithin the Ig receptor superfamily.

The results of a 200 gene mRNA Northern blot analysis is shown in FIG.18. The data depicted in FIG. 18 demonstrates, first, that the 200 geneproduces a transcript of approximately 1.2 kb in length, and, second,illustrates the TH1 specificity of 200 gene expression

For the study, three TH1 clones (D1.1, Dorris, AE7) and three TH2 clones(D10G.4, DAX, CDC25) were utilized, and RNA samples were isolated fromeither unstimulated cells (-) or cells which had been stimulated for 6hours with plate-bound anti-CD3 antibody (+). The samples were probedwith 200 gene sequences, and, as shown in FIG. 18, RNA from bothstimulated and unstimulated TH1 cells contained 200 gene mRNA, whilenone of the samples obtained from TH2 cells contained 200 gene mRNA. Itshould also be noted that 200 gene expression was higher in each of thestimulated TH1 cells relative to the corresponding unstimulated TH1cells.

As shown in Table 1, above, the 54 gene is expressed in a TH1-restrictedmanner. The 54 gene was identified via T cell paradigm searches in whichthe expression pattern of a TH1 cell clone, AE7, was compared to that ofa TH2 cell clone, D10.G4. The initial differential expression analysiswas performed using differential display techniques, as described,above, in Section 6.1.

The TH1-restricted pattern of the 54 gene expression was corroboratedthrough Northern analysis of RNA isolated from TH1 cell lines (AE7,D1.1, Dorris) and TH2 cell lines (D10.G4, DAX, CDC25), as shown in FIG.19. The TH1/TH2 Northern blot data depicted in FIG. 19 additionallyillustrates 54 gene expression within cell clones either stimulated orunstimulated with anti-CD3 antibodies, and demonstrates that 54 geneexpression goes down within stimulated TH1 cells.

To further characterize the 54 gene expression, a detailed time coursestudy was conducted using RNA isolated from AE7 clones. Specifically,RNA was isolated from unstimulated AE7 clones as well as from AE7 cloneswhich had been stimulated with anti-CD3 antibodies for varying lengthsof time, as noted in FIG. 20. As illustrated in FIG. 20, 54 geneexpression decreased slightly by 2-6 hours after stimulation and had notagain achieved pre-stimulation levels within 48 hours after stimulation.

A 54 gene expression analysis of cell lines representing a variety of Tcells, B cells and monocytic/macrophage cell lines was performed whichfailed to detect 54 gene expression in non-TH1 cells, demonstrating that54 gene expression is highly restricted to TH1-like cells. A Northernanalysis of 54 gene expression within tissues (FIG. 21), alsodemonstrated an expression profile consistent with that of a TH1cell-restricted expression profile. Namely, as shown in FIG. 21, mostorgans failed to express the 54 gene, while the highest level of 54 geneexpression was seen in lymph node tissue, and lowest detectable level ofexpression was seen in spleen, testis and uterus.

Band 54 nucleotide sequence, which had been obtained from the amplifiedcDNA produced in the initial differential display analysis in which the54 gene was identified, was used to isolate seven cDNA clones,designated 54A-G. Each of the clones were of similar size. The 54-C cDNAhas been deposited with the NRRL within the E. coli clone, 54-C.

FIG. 22 shows the entire 54 gene coding sequence (SEQ ID NO:11). Thederived amino acid sequence of the 54 gene product is also shown in FIG.22 (SEQ ID NO:12). Based on database homology searches, the 54 geneappears to encode a novel cysteine protease. Cysteine proteases areenzymes which contribute to intracellular protein degradation and appearto play a role in tissue degradation. It is possible, therefore, thatthe inhibition of 54 gene expression and/or 54 gene product activity inimmune disorders involving TH1-like cells may serve to minimize anytissue damage.

FIG. 23 depicts the 54 gene product amino acid sequence and points outsome of its potential cysteine protease-like features. For example, the54 gene product has an amino terminal end which resembles a cysteineprotease preproenzyme region, which is cleaved away upon formation ofthe active cysteine protease. The boxed region, from amino acid residue56 to 75 represents an "ERFNIN" region which has previously been notedas a feature of several cysteine proteases (Ishidoh, K. et al.,1987,)/FEBS Lett. 226:33-37). The circled amino acid residues within theboxed region represent conserved amino acid residues. The individualboxed amino acid residues represent residues that, based on homology,are thought to lie within the active site of the enzyme.

9. Example: Production of a 200 Gene Product-Ig Fusion

In this Section, the construction of a soluble Ig-fusion proteincontaining 200 gene product extracellular domain sequences is described.

The 200 gene product extracellular domain sequence of the Ig-fusionprotein consists of amino acid residues 1 to 191 (i.e., the 200 geneproduct portion ends with the amino acid sequence G-E-T-I-R-T). This 200gene product extracellular domain is PCR amplified using syntheticoligonucleotides complementary to the 200 gene nucleotide sequence suchthat the PCR products will include the above 200 gene product amino acidresidues 1-191. Further, XhoI and BamHI restriction sites are designedat the 5' and 3' ends of the PCR products, respectively, to facilitatethe subsequent insertion into IgG1 expression vectors (See Arurro, A. etal., 1991, Cell 61:1303-1313). In addition to the Ig-region encoded bythe IgG1 vectors, the Fc portion of the Ig region can be modified, byamino acid substitutions, to reduce complement activation and Fcbinding. (See, e.g., European Patent No. 239400 B1, Aug. 3, 1994).

PCR reaction conditions consist of 25 cycles of 30 sec. at 95° C., 30sec. at 60° C., and 30 sec. at 72° C. using Vent DNA polymerase (NewEngland Biolabs, Beverly, Mass.) and the 200 gene as a template.

The 5' oligonucleotide contains sequence upstream of the translationstart codon and, additionally, includes an XhoI site, as follows: 5'-AAATTT ATT CTC GAG GAC CCA CGC GTC CGG ATT TC-3' (SEQ ID NO:22).

The 3'-PCR oligonucleotide primer is complementary to the end of the 200gene extracellular domain and, additionally, includes a BamHI site,follows: 5'-TTA ATT TGG GAT CCC AGT TCT GAT CGT TTC TCC AGA GTC-3' (SEQID NO:23).

The 200 gene-Ig fusion construct can be expressed in a variety of cells,including COS, E. coli and baculovirus systems.

10. Deposit of Microorganisms

The following microorganisms were deposited under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure, and comply with thecriteria set forth in 37 CFR § 1.801-1.809, assurances regardingavailability and permanency of deposits. The microorganisms weredeposited with the Agricultural Research Service Culture Collection(NRRL) 1815 N. University Street, Peoria, Ill., on Jan. 19, 1995 (10-C,57-E, 105-A, 106-H, 161-G, 200-O), Mar. 3, 1995 (E. coli DH10B(Zip)™containing 200-P) and Jun. 6, 1995 (200-AF, 10-X, 54-C) and assigned theindicated accession numbers:

    ______________________________________                                        Microorganism   NRRL Accession No.                                            ______________________________________                                        10-C            B-21390                                                         57-E B-21391                                                                  105-A B-21392                                                                 106-H B-21393                                                                 161-G B-21394                                                                 200-O B-21395                                                                 E. coli  B-21416                                                              DH10B (Zip) ™                                                              containing 200-P cDNA                                                         200-AF B-21415                                                                10-X B-21455                                                                  54-C B-21456                                                                ______________________________________                                    

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 24                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 357 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTGGTGAGGG GGATCTACAA CTTGTTCGGT TAAAGAAAAA AGCAACAGCC AA -            #CAGAAATG     60                                                                 - - TGGTTATCCT TCACCTACCT AAAAAGGGAG ATGATGTGAA ACCAGGAACC AG -            #ATGCCGAG    120                                                                 - - TAGCAGGATG GGGGAGATTT GGCAATAAGT CAGCTCCCTC TGAAACTCTG AG -            #AGAAGTCA    180                                                                 - - ACATCACTGT CATAGACAGA AAAATCTGCA ATGATGAAAA ACACTATAAT TT -            #TCATCCTG    240                                                                 - - TAATTGGTCT AAACATGATT TGGGCAGGGG ACCTCCCCGG CGGAAAGGAC TC -            #CTGCAATG    300                                                                 - - GGGATTCTGG CAGCCCTCTC CTATGTGATT GGTATTTGGG AAGCATCACC TC - #CTTTT           357                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 255 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TTAGCGCCAT TGCCATAGAG AGACCTCAGC CATCAATCAC TAGCACATGA TT -             #GACAGACA     60                                                                 - - GAGAATGGGA CTTTGGGCTT TGGCAATTCT GACACTTCCC ATGTATTTGA CA -            #GTTACGGA    120                                                                 - - GGGCAGTAAA TCGTCCTGGG GTCTGGAAAA TGAGGCTTTA ATTGTGAGAT GC -            #CCCCAAAG    180                                                                 - - AGGACGCTCG ACTTATCCTG TGGAATGGTA TTACTCAGAT ACAAATGAAA GT -            #ATTCCTAC    240                                                                 - - CCAAAAAAAA AAAAA              - #                  - #                      - #   255                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2055 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 496..1509                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CCGGGTCGAC CCACGCGTCC GAGCCTCCTC AGTCAAGAGA AGCATCCCTC CA -             #GAAACAGG     60                                                                 - - GAAACATGAC ACTTTTGAAA GAATGCCAAA CGGCGTGAAA ATAAAAACAG AG -            #CATTCCCA    120                                                                 - - TTTGCACCGA CCAATCTCCA ATCTCCTGTA AGATTCAAAA GGGCAAGCAA GA -            #GGCGGTGA    180                                                                 - - CCGTTCACGA AAGCTAAAAT CCCATGCTAT TGAACATGAA GACTTCTGAT GC -            #TTAAATCT    240                                                                 - - CATTAACTGC TTTAAGTCAC TCCCAGGAGC TTGGATCCCA ACTTCTAGCA GT -            #AATAGTCT    300                                                                 - - GTGTAAAAAA AAAAAAAAAA TCAGTCTACA ACCACTCTCT AAATGCATGG AT -            #GAACTCAT    360                                                                 - - CAGAACATCA AAACCCAAGG AAACCCTAAG AGAGAAGAAT TCTAATAAAA AG -            #AATTTTAC    420                                                                 - - ATTGAAAACT TACAAGGCAA GGTCCCTTTC CCTGCTGACA GCCTAAGAAG TG -            #ATGTAACT    480                                                                 - - GCCACTGTGA AGACC ATG GCG ATG AAC AGC ATG TGC - #ATT GAA GAG CAG        CGC     531                                                                                      Met - #Ala Met Asn Ser Met Cys Ile Glu Glu Gln A - #rg                        - # 1               5  - #                10                 - - CAC CTC GAA CAC TAT TTG TTC CCG GTG GTC TA - #C ATA ATT GTG TTT ATA          579                                                                       His Leu Glu His Tyr Leu Phe Pro Val Val Ty - #r Ile Ile Val Phe Ile                    15         - #         20         - #         25                      - - GTC AGC GTC CCA GCC AAC ATC GGA TCT TTA TG - #C GTA TCC TTT CTG CAA          627                                                                       Val Ser Val Pro Ala Asn Ile Gly Ser Leu Cy - #s Val Ser Phe Leu Gln                30             - #     35             - #     40                          - - GCG AAG AAG GAA AAT GAG CTA GGG ATT TAC CT - #C TTC AGT CTG TCC CTG          675                                                                       Ala Lys Lys Glu Asn Glu Leu Gly Ile Tyr Le - #u Phe Ser Leu Ser Leu            45                 - # 50                 - # 55                 - # 60       - - TCA GAC CTG CTG TAT GCG CTG ACT CTG CCC CT - #C TGG ATC AAT TAC ACT          723                                                                       Ser Asp Leu Leu Tyr Ala Leu Thr Leu Pro Le - #u Trp Ile Asn Tyr Thr                            65 - #                 70 - #                 75              - - TGG AAT AAA GAC AAC TGG ACT TTC TCT CCC AC - #C TTG TGC AAA GGA AGC          771                                                                       Trp Asn Lys Asp Asn Trp Thr Phe Ser Pro Th - #r Leu Cys Lys Gly Ser                        80     - #             85     - #             90                  - - GTT TTC TTC ACC TAC ATG AAC TTT TAC AGC AG - #C ACG GCG TTC CTC ACT          819                                                                       Val Phe Phe Thr Tyr Met Asn Phe Tyr Ser Se - #r Thr Ala Phe Leu Thr                    95         - #        100         - #        105                      - - TGC ATT GCC CTG GAC CGC TAT TTA GCA GTC GT - #C TAC CCT CTG AAG TTT          867                                                                       Cys Ile Ala Leu Asp Arg Tyr Leu Ala Val Va - #l Tyr Pro Leu Lys Phe               110              - #   115              - #   120                          - - TCC TTC CTA AGA ACG AGA AGA TTC GCG TTT AT - #T ACC AGC CTC TCC ATC          915                                                                       Ser Phe Leu Arg Thr Arg Arg Phe Ala Phe Il - #e Thr Ser Leu Ser Ile           125                 1 - #30                 1 - #35                 1 -      #40                                                                              - - TGG ATA TTA GAG TCC TTC TTT AAC TCT ATG CT - #T CTG TGG AAA GAT        GAA      963                                                                    Trp Ile Leu Glu Ser Phe Phe Asn Ser Met Le - #u Leu Trp Lys Asp Glu                          145  - #               150  - #               155              - - ACG AGT GTT GAA TAT TGT GAC TCG GAC AAA TC - #T AAT TTC ACT CTC TGC         1011                                                                       Thr Ser Val Glu Tyr Cys Asp Ser Asp Lys Se - #r Asn Phe Thr Leu Cys                       160      - #           165      - #           170                  - - TAT GAC AAA TAC CCT CTG GAG AAA TGG CAG AT - #A AAC CTC AAC CTG TTT         1059                                                                       Tyr Asp Lys Tyr Pro Leu Glu Lys Trp Gln Il - #e Asn Leu Asn Leu Phe                   175          - #       180          - #       185                      - - CGG ACG TGC ATG GGC TAC GCA ATA CCC TTG AT - #C ACC ATC ATG ATC TGC         1107                                                                       Arg Thr Cys Met Gly Tyr Ala Ile Pro Leu Il - #e Thr Ile Met Ile Cys               190              - #   195              - #   200                          - - AAC CAT AAA GTC TAC CGA GCT GTG CGG CAC AA - #C CAA GCC ACG GAA AAC         1155                                                                       Asn His Lys Val Tyr Arg Ala Val Arg His As - #n Gln Ala Thr Glu Asn           205                 2 - #10                 2 - #15                 2 -      #20                                                                              - - AGC GAG AAG AGA AGG ATC ATA AAG TTG CTT GC - #T AGC ATC ACG TTG        ACT     1203                                                                    Ser Glu Lys Arg Arg Ile Ile Lys Leu Leu Al - #a Ser Ile Thr Leu Thr                          225  - #               230  - #               235              - - TTC GTC CTA TGC TTT ACC CCC TTC CAC GTG AT - #G GTG CTC ATC CGC TGC         1251                                                                       Phe Val Leu Cys Phe Thr Pro Phe His Val Me - #t Val Leu Ile Arg Cys                       240      - #           245      - #           250                  - - GTT TTA GAG CGC GAC ATG AAC GTC AAT GAC AA - #G TCT GGA TGG CAG ACG         1299                                                                       Val Leu Glu Arg Asp Met Asn Val Asn Asp Ly - #s Ser Gly Trp Gln Thr                   255          - #       260          - #       265                      - - TTT ACG GTG TAC AGA GTC ACA GTA GCC CTG AC - #G AGT CTA AAC TGT GTT         1347                                                                       Phe Thr Val Tyr Arg Val Thr Val Ala Leu Th - #r Ser Leu Asn Cys Val               270              - #   275              - #   280                          - - GCC GAT CCC ATT CTG TAC TGC TTT GTG ACT GA - #G ACG GGG AGA GCT GAT         1395                                                                       Ala Asp Pro Ile Leu Tyr Cys Phe Val Thr Gl - #u Thr Gly Arg Ala Asp           285                 2 - #90                 2 - #95                 3 -      #00                                                                              - - ATG TGG AAC ATA TTA AAA TTG TGT ACT AGG AA - #A CAC AAT AGA CAC        CAA     1443                                                                    Met Trp Asn Ile Leu Lys Leu Cys Thr Arg Ly - #s His Asn Arg His Gln                          305  - #               310  - #               315              - - GGG AAA AAA AGG GAC ATA CTT TCT GTG TCC AC - #A AGA GAT GCT GTA GAA         1491                                                                       Gly Lys Lys Arg Asp Ile Leu Ser Val Ser Th - #r Arg Asp Ala Val Glu                       320      - #           325      - #           330                  - - TTA GAG ATT ATA GAC TAA GAGGTGGAGG CAGGTTAAGT TA - #CATGGTAT                1539                                                                       Leu Glu Ile Ile Asp                                                                   335                                                                    - - TATTTAATGA AACTTACATT TTGGAAAAGA AATCTGGCAT AGTAGAACCC AG -             #TGGAAATA   1599                                                                 - - GTTTGAAGGT ACATTGTATG ACTCCTATGT TGGCTTTATT AAGTAAGGTA TA -            #GAAATGTA   1659                                                                 - - TTATCTTGTA TGTATTCTAA TGACTAGGCA TCATTGTTTT AGTACCAATT CT -            #CTTTGCCT   1719                                                                 - - CTATGTTATA ACCCCTAAGA AGCACGCGGG ACTGTTCGTC TTTAAATCAG TG -            #GCCATTCT   1779                                                                 - - ATCTGACTAC TATGACTTTT TGTTGTTGTT CTGCTTTGGG TTTTCAGTCT GC -            #CTGCATCA   1839                                                                 - - GTCTTCTCCT CTGTATACGT CTGTCTTCAA CAAATGTAAG GACTAAATAC CC -            #CTCCCGAT   1899                                                                 - - CACATCCATT ATCAAGGATT TGAAGCCACT CCATGTACTG GGTTATAAAA GA -            #AATGTTCT   1959                                                                 - - CATGAACTTT CATGAAGTTT ACATACCTTT GGGGATCTAG TCACCGAGTC AC -            #ATAAAGTA   2019                                                                 - - AAAGTAAATG GAAAAAAAAA AAAAAAAAAA AAGGGC      - #                       - #     2055                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 460 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CGCCAGTGTG CTGGAATTCG GCTTAGAGCA TTTCTTTCAA ACCACAGGTT AA -            #CACACACT     60                                                                 - - TACTAAAAAG CAATGCTGTT AGAGGAGAAG GGCTTGGGAG ACTCGGCCAT TT -            #GAAACANA    120                                                                 - - AGCAAGGCAC TCTCCAGGNN CAGCAAGTGG ATTCCCATTT CCTGCTGAGG GC -            #GGGTTCAC    180                                                                 - - ACTGAGACTG CACTCCAGTC AGCGGGAGGA ATCACCTGCA TTAATGCTTG TC -            #CTCTGCAG    240                                                                 - - AGCTAGTGTG CCTTCCACTC TGGGTACACT TGGGTGTCAA CATTTCAAAA TG -            #ATGACCTA    300                                                                 - - AGAGGCTCTC ATAGTTGGTG ATAACTATGG NAGGACAGAA GAACACTGGC TG -            #TATTGTCT    360                                                                 - - TTTTCTTTCA GCACTAGTGT CTTGGCCCTT AACTAAAACG GGTTCCATCA TC -            #CTCCAAAC    420                                                                 - - CAGGAAGATA GATTGTTAGA CAGGTCCTTT CCCCTCAACT     - #                      - #   460                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 414 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TTTTTTTTTT TNGGGAGAGG CTAGCACTGA AATTACAGTT TCAGTGGAAT TT -             #AGAGAAGT     60                                                                 - - AATAACTGCA AAAATTTATT TACACACACA CACACACACA CAGGGCATTT TA -            #CCTGTGTA    120                                                                 - - AGTGCAGTTT AATCANCCCC ATTACCTTAT GACCTTGGTT GGCAATGTCT CT -            #AAAGCTTT    180                                                                 - - AAAATTAAAA TAAAATTAAA AAGATGGTTT TCCATCTCAT AAAATCCCCT TT -            #GGGAATGG    240                                                                 - - AAGACTTCCT CTTTGGGGTN TTTTTTAGAG GGAACAGGAG GTAACTGTTA AT -            #TATTTATA    300                                                                 - - CATTCTAATA AACCATGAAT GCACCACATA AAATACTGTA CTCGGGGAGC AA -            #ACACTGTN    360                                                                 - - TGGGGGGGTT CTCTCTTACC AGAAGGAACA GGGGGCTTTT CAATGGCTGT GG - #GC              414                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 240 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - TTTNNGGGAC AGGGTTTCNC TGTGTATCTC TGGCTGTCCT GGAACTNACT CT -             #GTAGACCA     60                                                                 - - GGTTGGCCTC GANCTCAGAA ATCTACCTGC CTCTCCCTCC ANAGTGCTGG GA -            #TTAANGGT    120                                                                 - - GTATGCCACC AATNCCCGGC CTTAATATAT TNNTAAACAA CTTCATTTGA AT -            #GANATATT    180                                                                 - - GACACTACCC TTGGAATAAG AGTNCCCAGA ATGANGTACA GGNTTCANGG AA -            #TCATTTAA    240                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 217 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CTTAGCAGGT GGAGTTGCAG CAGGAAGCCT GGTAGCCACA CTCCAATCAG CA -            #GGGGTCCT     60                                                                 - - TGGACTCTCC ACATCAACAA ATGCCATCCT AGGGGCTGCT GGGGCACTGT TG -            #GAGCCTTG    120                                                                 - - CTCTGAGCTT AGGAGATGAC ACTTCTATCA GCTCAACTCA AAGCCTGTAC AG -            #ACTACGCA    180                                                                 - - GGAGATGAAG TTCCAAAAGG CACCTTCAGA ACCCTCA      - #                      - #     217                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2710 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 40..885                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - NGTCGACCCA CGCGTCCGGA TTTCCCCTCC CAAGTACTC ATG TTT TCA - # GGT CTT            54                                                                                         - #                  - #       Met Phe Ser Gly Leu                            - #                  - #         1         - #      5        - - ACC CTC AAC TGT GTC CTG CTG CTG CTG CAA CT - #A CTA CTT GCA AGG TCA          102                                                                       Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Le - #u Leu Leu Ala Arg Ser                            10 - #                 15 - #                 20              - - TTG GAA GAT GGT TAT AAG GTT GAG GTT GGT AA - #A AAT GCC TAT CTG CCC          150                                                                       Leu Glu Asp Gly Tyr Lys Val Glu Val Gly Ly - #s Asn Ala Tyr Leu Pro                        25     - #             30     - #             35                  - - TGC AGT TAC ACT CTA CCT ACA TCT GGG ACA CT - #T GTG CCT ATG TGC TGG          198                                                                       Cys Ser Tyr Thr Leu Pro Thr Ser Gly Thr Le - #u Val Pro Met Cys Trp                    40         - #         45         - #         50                      - - GGC AAG GGA TTC TGT CCT TGG TCA CAG TGT AC - #C AAT GAG TTG CTC AGA          246                                                                       Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Th - #r Asn Glu Leu Leu Arg                55             - #     60             - #     65                          - - ACT GAT GAA AGA AAT GTG ACA TAT CAG AAA TC - #C AGC AGA TAC CAG CTA          294                                                                       Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Se - #r Ser Arg Tyr Gln Leu            70                 - # 75                 - # 80                 - # 85       - - AAG GGC GAT CTC AAC AAA GGA GAT GTG TCT CT - #G ATC ATA AAG AAT GTG          342                                                                       Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Le - #u Ile Ile Lys Asn Val                            90 - #                 95 - #                100              - - ACT CTG GAT GAC CAT GGG ACC TAC TGC TGC AG - #G ATA CAG TTC CCT GGT          390                                                                       Thr Leu Asp Asp His Gly Thr Tyr Cys Cys Ar - #g Ile Gln Phe Pro Gly                       105      - #           110      - #           115                  - - CTT ATG AAT GAT AAA AAA TTA GAA CTG AAA TT - #A GAC ATC AAA GCA GCC          438                                                                       Leu Met Asn Asp Lys Lys Leu Glu Leu Lys Le - #u Asp Ile Lys Ala Ala                   120          - #       125          - #       130                      - - AAG GTC ACT CCA GCT CAG ACT GCC CAT GGG GA - #C TCT ACT ACA GCT TCT          486                                                                       Lys Val Thr Pro Ala Gln Thr Ala His Gly As - #p Ser Thr Thr Ala Ser               135              - #   140              - #   145                          - - CCA AGA ACC CTA ACC ACG GAG AGA AAT GGT TC - #A GAG ACA CAG ACA CTG          534                                                                       Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Se - #r Glu Thr Gln Thr Leu           150                 1 - #55                 1 - #60                 1 -      #65                                                                              - - GTG ACC CTC CAT AAT AAC AAT GGA ACA AAA AT - #T TCC ACA TGG GCT        GAT      582                                                                    Val Thr Leu His Asn Asn Asn Gly Thr Lys Il - #e Ser Thr Trp Ala Asp                          170  - #               175  - #               180              - - GAA ATT AAG GAC TCT GGA GAA ACG ATC AGA AC - #T GCT ATC CAC ATT GGA          630                                                                       Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Th - #r Ala Ile His Ile Gly                       185      - #           190      - #           195                  - - GTG GGA GTC TCT GCT GGG TTG ACC CTG GCA CT - #T ATC ATT GGT GTC TTA          678                                                                       Val Gly Val Ser Ala Gly Leu Thr Leu Ala Le - #u Ile Ile Gly Val Leu                   200          - #       205          - #       210                      - - ATC CTT AAA TGG TAT TCC TGT AAG AAA AAG AA - #G TTA TCG AGT TTG AGC          726                                                                       Ile Leu Lys Trp Tyr Ser Cys Lys Lys Lys Ly - #s Leu Ser Ser Leu Ser               215              - #   220              - #   225                          - - CTT ATT ACA CTG GCC AAC TTG CCT CCA GGA GG - #G TTG GCA AAT GCA GGA          774                                                                       Leu Ile Thr Leu Ala Asn Leu Pro Pro Gly Gl - #y Leu Ala Asn Ala Gly           230                 2 - #35                 2 - #40                 2 -      #45                                                                              - - GCA GTC AGG ATT CGC TCT GAG GAA AAT ATC TA - #C ACC ATC GAG GAG        AAC      822                                                                    Ala Val Arg Ile Arg Ser Glu Glu Asn Ile Ty - #r Thr Ile Glu Glu Asn                          250  - #               255  - #               260              - - GTA TAT GAA GTG GAG AAT TCA AAT GAG TAC TA - #C TGC TAC GTC AAC AGC          870                                                                       Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr Ty - #r Cys Tyr Val Asn Ser                       265      - #           270      - #           275                  - - CAG CAG CCA TCC TGA CCGCCTCTGG ACTGCCACTT TTAAAGGCT - #C GCCTTCATTT          925                                                                       Gln Gln Pro Ser                                                                       280                                                                    - - CTGACTTTGG TATTTCCCTT TKTGGAAAAC TATGTGATAT GTCACTTGGC AA -             #CCTCATTG    985                                                                 - - GAGGTTCTGA CCACAGCCAC TGAGAAAAGA GTTCCAGTTT TCTGGGGATA AT -            #TAACTCAC   1045                                                                 - - AAGGGGATTC GACTGTAACT CATGCTACAT TGAAATGCTC CATTTTATCC CT -            #GAGTTTCA   1105                                                                 - - GGGATCGGAT CTCCCACTCC AGAGACTTCA ATCATGCGTG TTGAAGCTCA CT -            #CGTGCTTT   1165                                                                 - - CATACATTAG GAATGGTTAG TGTGATGTCT TTGAGACATA GAGGTTTGTG GT -            #ATATCCGC   1225                                                                 - - AAAGCTCCTG AACAGGTAGG GGGAATAAAG GGCTAAGATA GGAAGGTGCG GT -            #CTTTGTTG   1285                                                                 - - ATGTTGGAAA ATCTTAAAGA AGTTGGTAGC TTTTCTAGAG ATTTCTGACC TT -            #GAAAGATT   1345                                                                 - - AAGAAAAAGC CAGGTGGCAT ATGCTTAACA CGATATAACT TGGGAACCTT AG -            #GCAGGAGG   1405                                                                 - - GTGATAAGTT CAAGGTCAGC CAGGGCTATG CTGGTAAGAC TGTCTCAMCA TC -            #CAAAGACG   1465                                                                 - - AAAATAAACA TAGAGACAGC AGGAGGCTGG AGATGAGGCT CGGACAGTGA GG -            #TGCATTGT   1525                                                                 - - GTACAAGCAC GAGGAATCTA TATTTGATCG TAGACCCCAC ATGAAAAAGC TA -            #GGCCTGGT   1585                                                                 - - AGAGCATGCT TGTAGACTCA AGAGATGGAG AGGTAAAGGC ACAACAGATC CC -            #CGGGGCTT   1645                                                                 - - GCGTGCAGTC AGCTTAGCCT AGGTGCTGAG TTCCAAGTCC ACAAGAGTCC CT -            #GTCTCAMA   1705                                                                 - - GTAAGATGGR CTGAGTATCT GGCGCATGTC CATGGGGGTT GTCCTCTCCT CT -            #CAGAAGAG   1765                                                                 - - ACATGCACAT GWCCCTGCAC ACACACACAC ACACACACAC ACACACACAC AC -            #ACACACAC   1825                                                                 - - ACACATGAWA TGAAGGTTCT CTCTGTGCCT GCTACCTCTC TATAACATGT AT -            #CTCTACAG   1885                                                                 - - GACTCTCCTC TGCCTCTGTT AAGACATGAG TGGGAGCATG GCAGAGCAGT CC -            #AGTAATTT   1945                                                                 - - ATTCCAGCAC TCAGAAGGCT GGAGCAGAAG CGTGGAGAGT TCAGGAGCAC TG -            #TGCCCAAC   2005                                                                 - - ACTGCCAGAC TCTTCTTACA CAAGAAAAAG GTTACCCGCA AGCAGCCTGC TG -            #TCTGTAAA   2065                                                                 - - AGGAAACCCT GCGAAAGGCA AACTTTGACT GTTGTGTGCT CAAGGGGAAC TG -            #ACTCAGAC   2125                                                                 - - AACTTCTCCA TTCCTGGAGG AAACTGGAGC TGTTTCTGAC AGAAGAACAA CC -            #GGTGACTG   2185                                                                 - - GGACATACGA AGGCAGAGCT CTTGCAGCAA TCTATATAGT CAGCAAAATA TT -            #CTTTGGGA   2245                                                                 - - GGACAGTCGT CACCAAATTG ATTTCCAAGC CGGTGGACCT CAGTTTCATC TG -            #GCTTACAG   2305                                                                 - - CTGCCTGCCC AGTGCCCTTG ATCTGTGCTG GCTCCCATCT ATAACAGAAT CA -            #AATTAAAT   2365                                                                 - - AGACCCCGAG TGAAAATATT AAGTGAGCAG AAAGGTAGCT TTGTTCAAAG AT -            #TTTTTTGC   2425                                                                 - - ATTGGGGAGC AACTGTGTAC ATCAGAGGAC ATCTGTTAGT GAGGACACCA AA -            #ACCTGTGG   2485                                                                 - - TACCGTTTTT TCATGTATGA ATTTTGTTGT TTAGGTTGCT TCTAGCTAGC TG -            #TGGAGGTC   2545                                                                 - - CTGGCTTTCT TAGGTGGGTA TGGAAGGGAG ACCATCTAAC AAAATCCATT AG -            #AGATAACA   2605                                                                 - - GCTCTCATGC AGAAGGGAAA ACTAATCTCA AATGTTTTAA AGTAATAAAA CT -            #GTACTGGC   2665                                                                 - - AAAGTACTTT GAGCATAAAA AAAAAAAAAA AAAAAGGGCG GCCGC   - #                    2710                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 337 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Met Ala Met Asn Ser Met Cys Ile Glu Glu Gl - #n Arg His Leu Glu His       1               5  - #                10  - #                15               - - Tyr Leu Phe Pro Val Val Tyr Ile Ile Val Ph - #e Ile Val Ser Val Pro                  20      - #            25      - #            30                   - - Ala Asn Ile Gly Ser Leu Cys Val Ser Phe Le - #u Gln Ala Lys Lys Glu              35          - #        40          - #        45                       - - Asn Glu Leu Gly Ile Tyr Leu Phe Ser Leu Se - #r Leu Ser Asp Leu Leu          50              - #    55              - #    60                           - - Tyr Ala Leu Thr Leu Pro Leu Trp Ile Asn Ty - #r Thr Trp Asn Lys Asp      65                  - #70                  - #75                  - #80        - - Asn Trp Thr Phe Ser Pro Thr Leu Cys Lys Gl - #y Ser Val Phe Phe Thr                      85  - #                90  - #                95               - - Tyr Met Asn Phe Tyr Ser Ser Thr Ala Phe Le - #u Thr Cys Ile Ala Leu                  100      - #           105      - #           110                  - - Asp Arg Tyr Leu Ala Val Val Tyr Pro Leu Ly - #s Phe Ser Phe Leu Arg              115          - #       120          - #       125                      - - Thr Arg Arg Phe Ala Phe Ile Thr Ser Leu Se - #r Ile Trp Ile Leu Glu          130              - #   135              - #   140                          - - Ser Phe Phe Asn Ser Met Leu Leu Trp Lys As - #p Glu Thr Ser Val Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Cys Asp Ser Asp Lys Ser Asn Phe Thr Le - #u Cys Tyr Asp Lys        Tyr                                                                                             165  - #               170  - #               175             - - Pro Leu Glu Lys Trp Gln Ile Asn Leu Asn Le - #u Phe Arg Thr Cys Met                  180      - #           185      - #           190                  - - Gly Tyr Ala Ile Pro Leu Ile Thr Ile Met Il - #e Cys Asn His Lys Val              195          - #       200          - #       205                      - - Tyr Arg Ala Val Arg His Asn Gln Ala Thr Gl - #u Asn Ser Glu Lys Arg          210              - #   215              - #   220                          - - Arg Ile Ile Lys Leu Leu Ala Ser Ile Thr Le - #u Thr Phe Val Leu Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Phe Thr Pro Phe His Val Met Val Leu Ile Ar - #g Cys Val Leu Glu        Arg                                                                                             245  - #               250  - #               255             - - Asp Met Asn Val Asn Asp Lys Ser Gly Trp Gl - #n Thr Phe Thr Val Tyr                  260      - #           265      - #           270                  - - Arg Val Thr Val Ala Leu Thr Ser Leu Asn Cy - #s Val Ala Asp Pro Ile              275          - #       280          - #       285                      - - Leu Tyr Cys Phe Val Thr Glu Thr Gly Arg Al - #a Asp Met Trp Asn Ile          290              - #   295              - #   300                          - - Leu Lys Leu Cys Thr Arg Lys His Asn Arg Hi - #s Gln Gly Lys Lys Arg      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Ile Leu Ser Val Ser Thr Arg Asp Ala Va - #l Glu Leu Glu Ile        Ile                                                                                             325  - #               330  - #               335             - - Asp                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 281 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Phe Ser Gly Leu Thr Leu Asn Cys Val Le - #u Leu Leu Leu Gln Leu        1               5 - #                 10 - #                 15              - - Leu Leu Ala Arg Ser Leu Glu Asp Gly Tyr Ly - #s Val Glu Val Gly Lys                   20     - #             25     - #             30                  - - Asn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pr - #o Thr Ser Gly Thr Leu               35         - #         40         - #         45                      - - Val Pro Met Cys Trp Gly Lys Gly Phe Cys Pr - #o Trp Ser Gln Cys Thr           50             - #     55             - #     60                          - - Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Va - #l Thr Tyr Gln Lys Ser       65                 - # 70                 - # 75                 - # 80       - - Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Ly - #s Gly Asp Val Ser Leu                       85 - #                 90 - #                 95              - - Ile Ile Lys Asn Val Thr Leu Asp Asp His Gl - #y Thr Tyr Cys Cys Arg                  100      - #           105      - #           110                  - - Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Ly - #s Leu Glu Leu Lys Leu              115          - #       120          - #       125                      - - Asp Ile Lys Ala Ala Lys Val Thr Pro Ala Gl - #n Thr Ala His Gly Asp          130              - #   135              - #   140                          - - Ser Thr Thr Ala Ser Pro Arg Thr Leu Thr Th - #r Glu Arg Asn Gly Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Thr Gln Thr Leu Val Thr Leu His Asn As - #n Asn Gly Thr Lys        Ile                                                                                             165  - #               170  - #               175             - - Ser Thr Trp Ala Asp Glu Ile Lys Asp Ser Gl - #y Glu Thr Ile Arg Thr                  180      - #           185      - #           190                  - - Ala Ile His Ile Gly Val Gly Val Ser Ala Gl - #y Leu Thr Leu Ala Leu              195          - #       200          - #       205                      - - Ile Ile Gly Val Leu Ile Leu Lys Trp Tyr Se - #r Cys Lys Lys Lys Lys          210              - #   215              - #   220                          - - Leu Ser Ser Leu Ser Leu Ile Thr Leu Ala As - #n Leu Pro Pro Gly Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Ala Asn Ala Gly Ala Val Arg Ile Arg Se - #r Glu Glu Asn Ile        Tyr                                                                                             245  - #               250  - #               255             - - Thr Ile Glu Glu Asn Val Tyr Glu Val Glu As - #n Ser Asn Glu Tyr Tyr                  260      - #           265      - #           270                  - - Cys Tyr Val Asn Ser Gln Gln Pro Ser                                              275          - #       280                                             - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1257 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 22..1137                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CCGGGTCGAC CCACGCGTCC G ATG ACA CTG ACT GCC CAC - #CTC TCC TAC TTT           51                                                                                          - #      Met Thr Leu Thr Ala His Leu - #Ser Tyr Phe                           - #        1          - #     5             - #     10       - - CTG GTC CTG TTG TTA GCG GGC CAA GGC CTC AG - #T GAC TCC CTC CTC ACC           99                                                                       Leu Val Leu Leu Leu Ala Gly Gln Gly Leu Se - #r Asp Ser Leu Leu Thr                            15 - #                 20 - #                 25              - - AAG GAT GCA GGT CCC CGC CCA CTG GAG CTG AA - #G GAA GTC TTC AAG CTG          147                                                                       Lys Asp Ala Gly Pro Arg Pro Leu Glu Leu Ly - #s Glu Val Phe Lys Leu                        30     - #             35     - #             40                  - - TTC CAG ATC CGG TTC AAC CGG AGT TAC TGG AA - #C CCA GCA GAG TAC ACT          195                                                                       Phe Gln Ile Arg Phe Asn Arg Ser Tyr Trp As - #n Pro Ala Glu Tyr Thr                    45         - #         50         - #         55                      - - CGC CGT CTG AGC ATC TTT GCC CAC AAT CTG GC - #T CAG GCT CAA AGG CTA          243                                                                       Arg Arg Leu Ser Ile Phe Ala His Asn Leu Al - #a Gln Ala Gln Arg Leu                60             - #     65             - #     70                          - - CAG CAA GAA GAC TTG GGT ACA GCT GAG TTT GG - #A GAG ACT CCA TTC AGT          291                                                                       Gln Gln Glu Asp Leu Gly Thr Ala Glu Phe Gl - #y Glu Thr Pro Phe Ser            75                 - # 80                 - # 85                 - # 90       - - GAC CTC ACA GAG GAG GAG TTT GGC CAG TTA TA - #C GGG CAG GAG AGG TCA          339                                                                       Asp Leu Thr Glu Glu Glu Phe Gly Gln Leu Ty - #r Gly Gln Glu Arg Ser                            95 - #                100 - #                105              - - CCA GAA AGG ACC CCC AAC ATG ACC AAA AAG GT - #A GAG TCT AAC ACG TGG          387                                                                       Pro Glu Arg Thr Pro Asn Met Thr Lys Lys Va - #l Glu Ser Asn Thr Trp                       110      - #           115      - #           120                  - - GGG GAA TCT GTG CCC CGC ACC TGT GAC TGG CG - #T AAA GCA AAG AAC ATC          435                                                                       Gly Glu Ser Val Pro Arg Thr Cys Asp Trp Ar - #g Lys Ala Lys Asn Ile                   125          - #       130          - #       135                      - - ATC TCG TCG GTC AAG AAC CAG GGA AGC TGC AA - #A TGC TGC TGG GCC ATG          483                                                                       Ile Ser Ser Val Lys Asn Gln Gly Ser Cys Ly - #s Cys Cys Trp Ala Met               140              - #   145              - #   150                          - - GCA GCT GCC GAC AAC ATC CAG GCT CTG TGG CG - #C ATC AAA CAC CAG CAG          531                                                                       Ala Ala Ala Asp Asn Ile Gln Ala Leu Trp Ar - #g Ile Lys His Gln Gln           155                 1 - #60                 1 - #65                 1 -      #70                                                                              - - TTT GTG GAC GTC TCT GTG CAG GAG CTG CTG GA - #C TGC GAA CGC TGT        GGA      579                                                                    Phe Val Asp Val Ser Val Gln Glu Leu Leu As - #p Cys Glu Arg Cys Gly                          175  - #               180  - #               185              - - AAT GGT TGC AAT GGT GGC TTC GTG TGG GAC GC - #A TAT CTA ACT GTC CTC          627                                                                       Asn Gly Cys Asn Gly Gly Phe Val Trp Asp Al - #a Tyr Leu Thr Val Leu                       190      - #           195      - #           200                  - - AAC AAC AGT GGC CTG GCC AGT GAA AAG GAT TA - #T CCA TTC CAG GGG GAC          675                                                                       Asn Asn Ser Gly Leu Ala Ser Glu Lys Asp Ty - #r Pro Phe Gln Gly Asp                   205          - #       210          - #       215                      - - AGA AAG CCT CAC AGA TGC CTA GCC AAG AAG TA - #C AAG AAG GTG GCC TGG          723                                                                       Arg Lys Pro His Arg Cys Leu Ala Lys Lys Ty - #r Lys Lys Val Ala Trp               220              - #   225              - #   230                          - - ATC CAG GAT TTC ACC ATG TTG TCC AAT AAT GA - #G CAG GCA ATT GCC CAC          771                                                                       Ile Gln Asp Phe Thr Met Leu Ser Asn Asn Gl - #u Gln Ala Ile Ala His           235                 2 - #40                 2 - #45                 2 -      #50                                                                              - - TAC CTG GCC GTG CAT GGA CCT ATC ACC GTG AC - #C ATC AAC ATG AAA        CTA      819                                                                    Tyr Leu Ala Val His Gly Pro Ile Thr Val Th - #r Ile Asn Met Lys Leu                          255  - #               260  - #               265              - - CTC CAG CAT TAC CAG AAG GGT GTC ATC AAG GC - #T ACA CCC AGC TCC TGT          867                                                                       Leu Gln His Tyr Gln Lys Gly Val Ile Lys Al - #a Thr Pro Ser Ser Cys                       270      - #           275      - #           280                  - - GAC CCT CGG CAA GTG GAC CAC TCT GTC TTG CT - #G GTG GGC TTT GGC AAG          915                                                                       Asp Pro Arg Gln Val Asp His Ser Val Leu Le - #u Val Gly Phe Gly Lys                   285          - #       290          - #       295                      - - GAG AAA GAG GGC ATG CAG ACA GGG ACA GTC TT - #G TCC CAT TCT CGA AAA          963                                                                       Glu Lys Glu Gly Met Gln Thr Gly Thr Val Le - #u Ser His Ser Arg Lys               300              - #   305              - #   310                          - - CGT CGC CAC TCC TCC CCA TAC TGG ATC CTG AA - #G AAC TCC TGG GGA GCT         1011                                                                       Arg Arg His Ser Ser Pro Tyr Trp Ile Leu Ly - #s Asn Ser Trp Gly Ala           315                 3 - #20                 3 - #25                 3 -      #30                                                                              - - CAC TGG GGC GAG AAG GGT TAC TTC AGG CTG TA - #T CGG GGA AAC AAC        ACC     1059                                                                    His Trp Gly Glu Lys Gly Tyr Phe Arg Leu Ty - #r Arg Gly Asn Asn Thr                          335  - #               340  - #               345              - - TGT GGA GTC ACC AAG TAT CCC TTC ACA GCT CA - #A GTG GAC TCA CCA GTA         1107                                                                       Cys Gly Val Thr Lys Tyr Pro Phe Thr Ala Gl - #n Val Asp Ser Pro Val                       350      - #           355      - #           360                  - - AAG AAG GCA CGG ACC TCT TGT CCT CCC TGA AG - #GCAGCAGV CACTCTTCTG           1157                                                                       Lys Lys Ala Arg Thr Ser Cys Pro Pro                                                   365          - #       370                                             - - CTTCTCCCAC ATGGCCACTG CCCCTTGTCA GCCCTGCCCA CATCCTCTCT GT -             #ATGGCTTC   1217                                                                 - - ATAAACCAAG ACTGCTCCGT GAAAAAAAAA AAAAAAAAAA     - #                      - #  1257                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 371 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met Thr Leu Thr Ala His Leu Ser Tyr Phe Le - #u Val Leu Leu Leu Ala        1               5 - #                 10 - #                 15              - - Gly Gln Gly Leu Ser Asp Ser Leu Leu Thr Ly - #s Asp Ala Gly Pro Arg                   20     - #             25     - #             30                  - - Pro Leu Glu Leu Lys Glu Val Phe Lys Leu Ph - #e Gln Ile Arg Phe Asn               35         - #         40         - #         45                      - - Arg Ser Tyr Trp Asn Pro Ala Glu Tyr Thr Ar - #g Arg Leu Ser Ile Phe           50             - #     55             - #     60                          - - Ala His Asn Leu Ala Gln Ala Gln Arg Leu Gl - #n Gln Glu Asp Leu Gly       65                 - # 70                 - # 75                 - # 80       - - Thr Ala Glu Phe Gly Glu Thr Pro Phe Ser As - #p Leu Thr Glu Glu Glu                       85 - #                 90 - #                 95              - - Phe Gly Gln Leu Tyr Gly Gln Glu Arg Ser Pr - #o Glu Arg Thr Pro Asn                  100      - #           105      - #           110                  - - Met Thr Lys Lys Val Glu Ser Asn Thr Trp Gl - #y Glu Ser Val Pro Arg              115          - #       120          - #       125                      - - Thr Cys Asp Trp Arg Lys Ala Lys Asn Ile Il - #e Ser Ser Val Lys Asn          130              - #   135              - #   140                          - - Gln Gly Ser Cys Lys Cys Cys Trp Ala Met Al - #a Ala Ala Asp Asn Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gln Ala Leu Trp Arg Ile Lys His Gln Gln Ph - #e Val Asp Val Ser        Val                                                                                             165  - #               170  - #               175             - - Gln Glu Leu Leu Asp Cys Glu Arg Cys Gly As - #n Gly Cys Asn Gly Gly                  180      - #           185      - #           190                  - - Phe Val Trp Asp Ala Tyr Leu Thr Val Leu As - #n Asn Ser Gly Leu Ala              195          - #       200          - #       205                      - - Ser Glu Lys Asp Tyr Pro Phe Gln Gly Asp Ar - #g Lys Pro His Arg Cys          210              - #   215              - #   220                          - - Leu Ala Lys Lys Tyr Lys Lys Val Ala Trp Il - #e Gln Asp Phe Thr Met      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Ser Asn Asn Glu Gln Ala Ile Ala His Ty - #r Leu Ala Val His        Gly                                                                                             245  - #               250  - #               255             - - Pro Ile Thr Val Thr Ile Asn Met Lys Leu Le - #u Gln His Tyr Gln Lys                  260      - #           265      - #           270                  - - Gly Val Ile Lys Ala Thr Pro Ser Ser Cys As - #p Pro Arg Gln Val Asp              275          - #       280          - #       285                      - - His Ser Val Leu Leu Val Gly Phe Gly Lys Gl - #u Lys Glu Gly Met Gln          290              - #   295              - #   300                          - - Thr Gly Thr Val Leu Ser His Ser Arg Lys Ar - #g Arg His Ser Ser Pro      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Tyr Trp Ile Leu Lys Asn Ser Trp Gly Ala Hi - #s Trp Gly Glu Lys        Gly                                                                                             325  - #               330  - #               335             - - Tyr Phe Arg Leu Tyr Arg Gly Asn Asn Thr Cy - #s Gly Val Thr Lys Tyr                  340      - #           345      - #           350                  - - Pro Phe Thr Ala Gln Val Asp Ser Pro Val Ly - #s Lys Ala Arg Thr Ser              355          - #       360          - #       365                      - - Cys Pro Pro                                                                  370                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Met Arg Gln Lys Ala Val Ser Leu Phe Leu Cy - #s Tyr Leu Leu Leu Phe      1               5   - #                10  - #                15               - - Thr Cys Ser Gly Val Glu Ala Gly Lys Lys Ly - #s Cys Ser Glu Ser Ser                  20      - #            25      - #            30                   - - Asp Ser Gly Ser Gly Phe Trp Lys Ala Leu Th - #r Phe Met Ala Val Gly              35          - #        40          - #        45                       - - Gly Gly Leu Ala Val Ala Gly Leu Pro Ala Le - #u Gly Phe Thr Gly Ala          50              - #    55              - #    60                           - - Gly Ile Ala Ala Asn Ser Val Ala Ala Ser Le - #u Met Ser Trp Ser Ala      65                  - #70                  - #75                  - #80        - - Ile Leu Asn Gly Gly Gly Val Pro Ala Gly Gl - #y Leu Val Ala Thr Leu                      85  - #                90  - #                95               - - Gln Ser Leu Gly Ala Gly Gly Ser Ser Val Il - #e Thr Gly Asn Ile Gly                  100      - #           105      - #           110                  - - Ala Leu Met Gly Tyr Ala Thr His Lys Tyr Le - #u Asp Ser Glu Glu Asp              115          - #       120          - #       125                      - - Glu Glu                                                                      130                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Met Arg Gln Lys Ala Val Ser Val Phe Leu Cy - #s Tyr Leu Leu Leu Phe      1               5   - #                10  - #                15               - - Thr Cys Ser Gly Val Glu Ala Gly Lys Lys Ly - #s Cys Ser Glu Ser Ser                  20      - #            25      - #            30                   - - Asp Ser Gly Ser Gly Phe Trp Lys Ala Leu Th - #r Phe Met Ala Val Gly              35          - #        40          - #        45                       - - Gly Gly Leu Ala Val Ala Gly Leu Pro Ala Le - #u Gly Phe Thr Gly Ala          50              - #    55              - #    60                           - - Gly Ile Ala Ala Asn Ser Val Ala Ala Ser Le - #u Met Ser Trp Ser Ala      65                  - #70                  - #75                  - #80        - - Ile Leu Asn Gly Gly Gly Val Pro Ala Gly Gl - #y Leu Val Ala Thr Leu                      85  - #                90  - #                95               - - Gln Ser Leu Gly Ala Gly Gly Ser Ser Val Va - #l Ile Gly Asn Ile Gly                  100      - #           105      - #           110                  - - Ala Leu Met Arg Tyr Ala Thr His Lys Tyr Le - #u Asp Ser Glu Glu Asp              115          - #       120          - #       125                      - - Glu Glu                                                                      130                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Val Glu Ala Gly Lys Lys Lys Cys Ser Glu Se - #r Ser Asp Ser Gly Ser      1               5   - #                10  - #                15               - - Gly Phe Trp Lys Ala Leu Thr Phe Met Ala Va - #l Gly Gly Gly Leu Ala                  20      - #            25      - #            30                   - - Val Ala Gly Leu Pro Ala Leu Gly Phe Thr Gl - #y Ala Gly Ile Ala Ala              35          - #        40          - #        45                       - - Asn Ser Val Ala Ala Ser Leu Met Ser Trp Se - #r Ala Ile Leu Asn Gly          50              - #    55              - #    60                           - - Gly Gly Val Pro Ala Gly Gly Leu Val Ala Th - #r Leu Gln Ser Leu Gly      65                  - #70                  - #75                  - #80        - - Ala Gly Gly Ser Ser Val Val Ile Gly Asn Il - #e Gly Ala Leu Met Gly                      85  - #                90  - #                95               - - Tyr Ala Thr His Lys Tyr Leu Asp Ser Glu Gl - #u Asp Glu Glu                          100      - #           105      - #           110                  - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Gly Lys Lys Lys Cys Ser Glu Ser Ser Asp Se - #r Gly Ser Gly Phe Trp      1               5   - #                10  - #                15               - - Lys Ala Leu Thr Phe Met Ala Val Gly Gly Gl - #y Leu Ala Val Ala Gly                  20      - #            25      - #            30                   - - Leu Pro Ala Leu Gly Phe Thr Gly Ala Gly Il - #e Ala Ala Asn Ser Val              35          - #        40          - #        45                       - - Ala Ala Ser Leu Met Ser Trp Ser Ala Ile Le - #u Asn Gly Gly Gly Val          50              - #    55              - #    60                           - - Pro Ala Gly Gly Leu Val Ala Thr Leu Gln Se - #r Leu Gly Ala Gly Gly      65                  - #70                  - #75                  - #80        - - Ser Ser Val Val Ile Gly Asn Ile Gly Ala Le - #u Met Gly Tyr Ala Thr                      85  - #                90  - #                95               - - His Lys Tyr Leu Asp Ser Glu Glu Asp Glu Gl - #u                                      100      - #           105                                         - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 122 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Met Glu Ala Ser Ala Leu Thr Ser Ser Ala Va - #l Thr Ser Val Ala Lys      1               5   - #                10  - #                15               - - Val Val Arg Val Ala Ser Gly Ser Ala Val Va - #l Leu Pro Leu Ala Arg                  20      - #            25      - #            30                   - - Ile Ala Thr Val Val Ile Gly Gly Val Val Al - #a Met Ala Ala Val Pro              35          - #        40          - #        45                       - - Met Val Leu Ser Ala Met Gly Phe Thr Ala Al - #a Gly Ile Ala Ser Ser          50              - #    55              - #    60                           - - Ser Ile Ala Ala Lys Met Met Ser Ala Ala Al - #a Ile Ala Asn Gly Gly      65                  - #70                  - #75                  - #80        - - Gly Val Ala Ser Gly Ser Leu Val Gly Thr Le - #u Gln Ser Leu Gly Ala                      85  - #                90  - #                95               - - Thr Gly Leu Ser Gly Leu Thr Lys Phe Ile Le - #u Gly Ser Ile Gly Ser                  100      - #           105      - #           110                  - - Ala Ile Ala Ala Val Ile Ala Arg Phe Tyr                                          115          - #       120                                             - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TTGCCATAGA GAGACCTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - TGCTGTCCAA TTATACAGG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - GAACACGGCA TTGTCACTAA CT           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CCTCATAGAT GGGCACTGTG T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - AAATTTATTC TCGAGGACCC ACGCGTCCGG ATTTC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - TTAATTTGGG ATCCCAGTTC TGATCGTTTC TCCAGAGTC      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Phe Phe Val Phe Leu Ala Gly Gly Val Ala Al - #a Gly Ser Leu Val Ala       1               5  - #                10  - #                15               - - Thr Leu Gln Ser Ala Gly Val Leu Gly Leu Se - #r Thr Ser Thr Asn Ala                  20      - #            25      - #            30                   - - Ile Leu Gly Ala Ala Gly Ala Leu Leu Glu Pr - #o Cys Ser Glu Leu Arg              35          - #        40          - #        45                       - - Arg                                                                     __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from The group consisting of:(a) thenucleotide sequence contained in SEQ ID NO:3, in a plasmid within E.coli clone 10-C as deposited with the NRRL (NRRL Accession No. B-21390)or in the cDNA insert contained within E. coli clone 10-X as depositedwith the NRRL (NRRL Accession No. B-21455); (b) the nucleotide sequencecontained in SEQ ID NO:4 or in a plasmid within E. coli clone 57-E asdeposited with the NRRL (NRRL, Accession No. B-21391); (c) thenucleotide sequence contained in SEQ ID NO:5 or in a plasmid within E.coli clone 105-A as deposited with the NRRL (NRRL Accession No.B-21392); (d) the nucleotide sequence contained in SEQ ID NO:6 or in aplasmid within E. coli clone 106-H as deposited with the NRRL (NRRLAccession No. B-21393); and (e) the nucleotide sequence contained in SEQID NO:7 or in a plasmid within E. coli clone 161-G as deposited with theNRRL (NRRL Accession No. B-21394).
 2. The nucleic acid molecule of claim1, wherein the nucleic acid molecule comprises the nucleotide sequencecontained in SEQ ID NO:3, in a plasmid within E. coli clone 10-C asdeposited with the NRRL (NRRL Accession No. B-21390) or in the cDNAinsert contained within E. coli clone 10-X as deposited with the NRRL(NRRL Accession No. B-21455).
 3. The nucleic acid molecule of claim 1,wherein the nucleic acid molecule comprises the nucleotide sequencecontained in SEQ ID NO:4 or in a plasmid within E. coli clone 57-E asdeposited with the NRRL (NRRL, Accession No. B-21391).
 4. The nucleicacid molecule of claim 1, wherein the nucleic acid molecule comprisesthe nucleotide sequence contained in SEQ ID NO:5 or in a plasmid withinE. coli clone 105-A as deposited with the NRRL (NRRL Accession No.B-21392).
 5. The nucleic acid molecule of claim 1, wherein the nucleicacid molecule comprises the nucleotide sequence contained in SEQ ID NO:6or in a plasmid within E. coli clone 106-H as deposited with the NRRL(NRRL Accession No. B-21393).
 6. The nucleic acid molecule of claim 1,wherein the nucleic acid molecule comprises the nucleotide sequencecontained in SEQ ID NO:7 or in a plasmid within E. coli clone 161-G asdeposited with the NRRL (NRRL Accession No. B-21394).
 7. An isolatednucleic acid molecule which hybridizes to the nucleic acid molecule ofclaim 1 under highly stringent conditions comprising hybridization in0.5 M NaHPO₄, 7% sodium dodecyl sulfate, 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C., wherein said isolated nucleicacid molecule encodes a gene product functionally equivalent to a geneproduct encoded by the nucleic acid molecule of claim
 1. 8. An isolatednucleic acid molecule which encodes an amino acid sequence encoded bythe nucleotide sequence of claim 1 or its complement.
 9. An isolatednucleic acid molecule comprising a nucleotide sequence which encodes theamino acid sequence depicted in FIG. 9 (SEQ ID NO:9).
 10. A geneticallyengineered host cell containing the nucleic acid of claim 1 or
 8. 11. Avector containing the nucleic acid of claim 1 or
 8. 12. An expressionvector containing the nucleic acid of claim 1, or 8 in operativeassociation with a nucleotide sequence regulatory element that controlsexpression of the nucleotide sequence in a host cell.
 13. A geneticallyengineered host cell containing the nucleic acid of claim 1, or 8 inoperative association with a nucleotide sequence regulatory element thatcontrols expression of the nucleotide sequence in the host cell.